Cell therapy:  a method and a composition for treating diabetes

ABSTRACT

A method of increasing beta-islet cells from pancreases of rabbits and a composition for transplantation of beta-islet cells isolated and cultured from rabbit pancreases to promote natural insulin production among diabetic patients.

CROSS-REFERENCE TO RELATED APPLICATIONS

This international application claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Patent Application No. 61/798,890, filedMar. 15, 2013, entitled CELL THERAPY: A METHOD AND A COMPOSITION FORTREATING DIABETES, the entirety of which is incorporated herein.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to a cell therapy method and a composition fortransplantation of beta-islet cells isolated and cultured from animalpancreases to promote natural insulin production among people withdiabetes.

2. Description of Related Art

Type 1 diabetes affects more than one million Americans. Type 1 diabetesis the most severe form of the disease, in which the body's immunesystem attacks insulin-producing cells required to keep blood sugar atnormal levels. It is known that extremely low blood sugar can result inseizures, impaired cognition, or unconsciousness. In the most severecases, the complications are not well controlled by insulin.

Ideally, replacing insulin-producing cells in the pancreas can freediabetics from lifelong insulin injections and effectively cure thedisease. The transplantation of these “islet” cells can now be done intwo ways, through a whole pancreas transplant or through a less invasiveand less costly process of injecting just the islet cells. Successfulpancreas transplantation has been demonstrated to be effective insignificantly improving the quality of life of people with diabetes,primarily by eliminating the need for exogenous insulin and frequentdaily blood glucose measurements (Pancreas Transplantation for Patientswith Type 1 Diabetes. Diabetes Care. 25 (Supplement 1): 5111. January2002). However, pancreas transplants require lifelong immunosuppressiontherapy to prevent rejection of the graft and potential recurrence ofthe autoimmune process that may destroy pancreatic islet cells.

The transplantation of beta-islet cells from donor pancreases has beenshown to promote natural insulin production among patients with type 1and type 2 diabetes (see Sperling, M. A. Type 1 Diabetes: Etiology andTreatment. Totowa, N.J.: Humana Press Inc. 2003. p. 529-552; InsulinTherapy. In: Edelman, S. V. and Henry, R. R. Diagnosis and Management ofType 2 Diabetes. Caddo, Okla.: Professional Communications, Inc. 2002.p. 121-148). Islet cell transplantation can be performed as apercutaneous minimally invasive procedure, in which islet cells areinfused into the liver via the portal vein. However, like othertransplant patients, islet recipients must take immune-suppressing drugsto prevent rejection of the foreign cells.

Xenografts or xenotransplants of islet cells derived from pork or beefhas been studied and shown as requires immunosuppression.

The ability of pre-cultured beta-cells of pancreases of newly-bornrabbits to survive and to actively function in organism of xenogeneicrecipient was demonstrated by us in experiments on rats withexperimentally induced Diabetes Mellitus. (Skaletsky N. N. and others.1994 {4}). Expressed and long-term (8 weeks—experiment term)anti-diabetic effect of xnotransplantation of cultures of islet cells asin cases of administering into abdominal cavity and spleen, and also incases of administering it into transverse abdominal muscle. Afterexperiments, histological tests were conducted that at the place ofintroduction of xenotransplants discovered islet cells with preservedstructure and without signs of cellular immune reaction. At the sametime, clear signs of regeneration of own beta-cells in pancreases ofanimal-recipients were detected. In addition to a well-definedsugar-reducing effect, a well-defined medical-prophylactics effect ofxenotransplantation of islet cells cultures on distinctive latecomplication of diabetes—Nephropathy,—was noted during experiments(Skaletskaya G. N. and others (2005 {4}).

RU 2135193 to Skaletsky at al. discloses obtaining beta islet cells fromnewborn rabbit pancreas and to transplantation methods. Beta cells areobtained by migration from pancreatic fragments in a culturing methodrequiring addition of serum to a culture medium.

Thus, despite of current developments, islet cells from differentsources which would not require immunosuppression are needed.

All references cited herein are incorporated herein by reference intheir entireties.

BRIEF SUMMARY OF THE INVENTION

The invention provides a composition comprising neo-islet cells isolatedfrom rabbit pancreases, wherein the neo-islet cells are isolated by amethod comprising: harvesting said pancreases of newborn rabbits andplacing pancreases in a salt solution comprising an antibiotic at atemperature of 4-10° C.; removing vessels and excretory ducts from theharvested pancreases; obtaining minced pancreatic micro fragments fromsaid pancreases; transferring the minced pancreatic micro fragments intoa receptacle container, wherein the receptacle container is adapted fora rotating device; incubating the minced pancreatic micro fragments in aserum free medium at temperature 36° C. to 37° C. for 5 to 12 days at 0%to 5% CO2 for a first incubation period, wherein the receptaclecontainer rotated by the rotating device at a constant speed of 2 to 6RPM; periodically replacing the serum free medium with fresh serum freemedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are beta-islet cells; and collectingneo-islet cells formed by association of free floating beta-islet cells.

The invention provides a composition comprising beta-islet cell clustersisolated from rabbit pancreases, wherein the beta-islet cell clustersare isolated by a method comprising: harvesting said pancreases ofnewborn rabbits and placing pancreases in a salt solution comprising anantibiotic at a temperature of 4-10° C.; removing vessels and excretoryducts from the harvested pancreases; obtaining minced pancreatic microfragments from said pancreases; transferring the minced pancreatic microfragments into a receptacle container, wherein the receptacle containeris adapted for a rotating device; incubating the minced pancreatic microfragments in a serum free medium at temperature 36° C. to 37° C. for 5to 12 days at 0% to 5% CO2, wherein the receptacle container rotated bythe rotating device at a constant speed of 2 to 6 RPM; periodicallyreplacing the serum free medium with fresh serum free medium andremoving spontaneously destroyed unwanted cells comprising exocrinecells and blood cells and elements of connective tissue until at least80% of remaining cells are beta-islet cells; collecting the beta-isletcell clusters; incubating the beta-islet cell clusters in a serum freemedium at temperature 18° C. to 28° C. for 4 to 10 days at 0% to 5% CO2;periodically replacing the serum free medium with fresh serum freemedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are beta-islet cells; and collecting thebeta-islet cell clusters.

The invention provides a composition comprising progenic cells andbeta-islet cells isolated from rabbit pancreases, wherein the progeniccells and beta-islet cells are isolated by a method comprising:harvesting said pancreases of newborn rabbits and placing pancreases ina salt solution comprising an antibiotic at a temperature of 4-10° C.;removing vessels and excretory ducts from the harvested pancreases;obtaining minced pancreatic micro fragments from said pancreases;transferring the minced pancreatic micro fragments into a receptaclecontainer, wherein the receptacle container is adapted for a rotatingdevice; incubating the minced pancreatic micro fragments in a serum freemedium at temperature 36° C. to 37° C. for 5 to 12 days at 0% to 5% CO2,wherein the receptacle container rotated by the rotating device at aconstant speed of 2 to 6 RPM; periodically replacing the serum freemedium with fresh serum free medium and removing spontaneously destroyedunwanted cells comprising exocrine cells and blood cells and elements ofconnective tissue until at least 80% of remaining cells are beta-isletcells; collecting residual tissues of micro fragments; incubating theresidual tissues of micro fragments in 7% rabbit serum medium attemperature 36° C. to 37.2° C. for 5 to 10 days at 0% to 5% CO2;periodically replacing the medium with fresh medium and removingspontaneously destroyed unwanted cells comprising exocrine cells andblood cells and elements of connective tissue until at least 80% ofremaining cells are progenic cells or beta-islet cells; and collectingthe progenic cells and beta-islet cells.

The invention provides a method of obtaining neo-islet cells isolatedfrom rabbit pancreases, wherein the method comprising: harvesting saidpancreases of newborn rabbits and placing pancreases in a salt solutioncomprising an antibiotic at a temperature of 4-10° C.; removing vesselsand excretory ducts from the harvested pancreases; obtaining mincedpancreatic micro fragments from said pancreases; transferring the mincedpancreatic micro fragments into a receptacle container, wherein thereceptacle container is adapted for a rotating device; incubating theminced pancreatic micro fragments in a serum free medium at temperature36° C. to 37° C. for 5 to 12 days at 0% to 5% CO2 for a first incubationperiod, wherein the receptacle container rotated by the rotating deviceat a constant speed of 2 to 6 RPM; periodically replacing the serum freemedium with fresh serum free medium and removing spontaneously destroyedunwanted cells comprising exocrine cells and blood cells and elements ofconnective tissue until at least 80% of remaining cells are beta-isletcells; and collecting neo-islet cells formed by association of freefloating beta-islet cells.

The invention provides a method of obtaining beta-islet cell clustersisolated from rabbit pancreases, wherein the method comprising:harvesting said pancreases of newborn rabbits and placing pancreases ina salt solution comprising an antibiotic at a temperature of 4-10° C.;removing vessels and excretory ducts from the harvested pancreases;obtaining minced pancreatic micro fragments from said pancreases;transferring the minced pancreatic micro fragments into a receptaclecontainer, wherein the receptacle container is adapted for a rotatingdevice; incubating the minced pancreatic micro fragments in a serum freemedium at temperature 36° C. to 37° C. for 5 to 12 days at 0% to 5% CO2,wherein the receptacle container rotated by the rotating device at aconstant speed of 2 to 6 RPM; periodically replacing the serum freemedium with fresh serum free medium and removing spontaneously destroyedunwanted cells comprising exocrine cells and blood cells and elements ofconnective tissue until at least 80% of remaining cells are beta-isletcells; collecting the beta-islet cell clusters; incubating thebeta-islet cell clusters in a serum free medium at temperature 18° C. to28° C. for 4 to 10 days at 0% to 5% CO2; periodically replacing theserum free medium with fresh serum free medium and removingspontaneously destroyed unwanted cells comprising exocrine cells andblood cells and elements of connective tissue until at least 80% ofremaining cells are beta-islet cells; and collecting the beta-islet cellclusters.

The invention provides a method of obtaining a composition comprisingprogenic cells and beta-islet cells isolated from rabbit pancreases,wherein the method comprising: harvesting said pancreases of newbornrabbits and placing pancreases in a salt solution comprising anantibiotic at a temperature of 4-10° C.; removing vessels and excretoryducts from the harvested pancreases; obtaining minced pancreatic microfragments from said pancreases; transferring the minced pancreatic microfragments into a receptacle container, wherein the receptacle containeris adapted for a rotating device; incubating the minced pancreatic microfragments in a serum free medium at temperature 36° C. to 37° C. for 5to 12 days at 0% to 5% CO2, wherein the receptacle container rotated bythe rotating device at a constant speed of 2 to 6 RPM; periodicallyreplacing the serum free medium with fresh serum free medium andremoving spontaneously destroyed unwanted cells comprising exocrinecells and blood cells and elements of connective tissue until at least80% of remaining cells are beta-islet cells; collecting residual tissuesof micro fragments; incubating the residual tissues of micro fragmentsin 7% rabbit serum medium at temperature 36° C. to 37.2° C. for 5 to 10days at 0% to 5% CO2; periodically replacing the medium with freshmedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are progenic cells or beta-islet cells;and collecting the progenic cells and beta-islet cells.

The invention provides a method of treating diabetes in a patient inneed of such treatment by administration of neo-islet cells isolatedfrom rabbit pancreases, wherein the neo-islet cells are isolated by amethod comprising: harvesting said pancreases of newborn rabbits andplacing pancreases in a salt solution comprising an antibiotic at atemperature of 4-10° C.; removing vessels and excretory ducts from theharvested pancreases; obtaining minced pancreatic micro fragments fromsaid pancreases; transferring the minced pancreatic micro fragments intoa receptacle container, wherein the receptacle container is adapted fora rotating device; incubating the minced pancreatic micro fragments in aserum free medium at temperature 36° C. to 37° C. for 5 to 12 days at 0%to 5% CO2 for a first incubation period, wherein the receptaclecontainer rotated by the rotating device at a constant speed of 2 to 6RPM; periodically replacing the serum free medium with fresh serum freemedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are beta-islet cells; and collectingneo-islet cells formed by association of free floating beta-islet cells;and further wherein the neo-islet cells are administered to the patientto treat diabetes. The invention provides for neo-islet cells fromrabbit pancreases for use as a treatment for diabetes. The inventionprovides for the use of neo-islet cells isolated from rabbit pancreasesto make a composition to treat diabetes.

The invention provides a method of treating diabetes in a patient inneed of such treatment by administration of beta-islet cell clustersisolated from rabbit pancreases, wherein the beta-islet cell clustersare isolated by a method comprising: harvesting said pancreases ofnewborn rabbits and placing pancreases in a salt solution comprising anantibiotic at a temperature of 4-10° C.; removing vessels and excretoryducts from the harvested pancreases; obtaining minced pancreatic microfragments from said pancreases; transferring the minced pancreatic microfragments into a receptacle container, wherein the receptacle containeris adapted for a rotating device; incubating the minced pancreatic microfragments in a serum free medium at temperature 36° C. to 37° C. for 5to 12 days at 0% to 5% CO2, wherein the receptacle container rotated bythe rotating device at a constant speed of 2 to 6 RPM; periodicallyreplacing the serum free medium with fresh serum free medium andremoving spontaneously destroyed unwanted cells comprising exocrinecells and blood cells and elements of connective tissue until at least80% of remaining cells are beta-islet cells; collecting the beta-isletcell clusters; incubating the beta-islet cell clusters in a serum freemedium at temperature 18° C. to 28° C. for 4 to 10 days at 0% to 5% CO2;periodically replacing the serum free medium with fresh serum freemedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are beta-islet cells; and collecting thebeta-islet cell clusters; and further wherein the beta-islet cellclusters are administered to the patient to treat diabetes. Theinvention provides for beta-islet cell clusters from rabbit pancreasesfor use as a treatment for diabetes. The invention provides for the useof beta-islet cell clusters isolated from rabbit pancreases to make acomposition to treat diabetes.

The invention provides a method of treating diabetes in a patient inneed of such treatment by administration of progenic cells andbeta-islet cells isolated from rabbit pancreases, wherein the progeniccells and beta-islet cells are isolated by a method comprising:harvesting said pancreases of newborn rabbits and placing pancreases ina salt solution comprising an antibiotic at a temperature of 4-10° C.;removing vessels and excretory ducts from the harvested pancreases;obtaining minced pancreatic micro fragments from said pancreases;transferring the minced pancreatic micro fragments into a receptaclecontainer, wherein the receptacle container is adapted for a rotatingdevice; incubating the minced pancreatic micro fragments in a serum freemedium at temperature 36° C. to 37° C. for 5 to 12 days at 0% to 5% CO2,wherein the receptacle container rotated by the rotating device at aconstant speed of 2 to 6 RPM; periodically replacing the serum freemedium with fresh serum free medium and removing spontaneously destroyedunwanted cells comprising exocrine cells and blood cells and elements ofconnective tissue until at least 80% of remaining cells are beta-isletcells; collecting residual tissues of micro fragments; incubating theresidual tissues of micro fragments in 7% rabbit serum medium attemperature 36° C. to 37.2° C. for 5 to 10 days at 0% to 5% CO2;periodically replacing the medium with fresh medium and removingspontaneously destroyed unwanted cells comprising exocrine cells andblood cells and elements of connective tissue until at least 80% ofremaining cells are progenic cells or beta-islet cells; and collectingthe progenic cells and beta-islet cells; and further wherein theprogenic cells and beta-islet cells are administered to the patient totreat diabetes. The invention provides for progenic cells and beta-isletcells from rabbit pancreases for use as a treatment for diabetes. Theinvention provides for the use of progenic cells and beta-islet cellsisolated from rabbit pancreases to make a composition to treat diabetes.

The invention provides a method wherein the patient has type I diabetes.The invention provides a method wherein the administration isintraperitoneal, parenteral, intravenous, intramuscular, subcutaneous,or intrathecal. The invention provides a method wherein theadministration is into liver through portal vein, directly intoparenchyma of the liver, or into spleen. Harvesting the pancreases ofnewborn rabbits and placing pancreases in a salt solution comprising anantibiotic at a temperature of 4-10° C.; removing vessels and excretoryducts from the harvested pancreases; obtaining minced pancreatic microfragments from said pancreases.

The micro fragments are transferred into a receptacle container, such asa medical or laboratory roller bottle, that is adapted for a rotatingdevice, with a serum-free medium. The rotating device is housed in anincubator.

The minced pancreatic micro fragments in the serum-free medium areincubated in the rotating device at a constant speed at a firstincubation temperature 36° C. to 37.2° C. (preferably 37° C.) for 5 to12 days (preferably 8-10 days) at 0% to 5% CO₂ for a first incubationperiod.

During the first incubation, free floating beta-islet cells associateinto more dense neo-islet cells that are spherical structures. Theneo-islet cells so obtained can be used directly in transplantation todiabetes patient without immunosuppressive therapy as the neo-beta isletcells comprise only non-immunogenic beta-islet cells.

Also formed during the first incubation period are clusters ofbeta-islet cells that are up to 1 mm in diameter. The beta-islet cellclusters are harvested and further incubated for a second incubationperiod. During the second incubation period, the following conditionsare maintained: a temperature of 18-28° C. (preferably 24° C.), aduration of 4-10 days (preferably 6-8 days), a serum-free medium (suchas medium 199), and 0-5% CO₂. During the second incubation period, theserum free medium is periodically (eg. every 3-4 days) replaced, allspontaneously destroyed unwanted cells comprising exocrine cells, bloodcells and elements of connective tissue are removed, and beta-islet cellclusters remained in at least 80%.

It is known that precursors of mature beta-cells are located inepithelium of fine excretory ducts of pancreases. These cells can beattributed to regional stem cells or progenic cells. As a source of suchcells we used tissues of micro fragments of pancreases of newbornrabbits that remained after the first incubation period described above.During these days, in general, death and elimination of exocrine cellsoccurs, as well as migration of beta cells. Remains of pancreaticfragments, comprising in general of fine excretory ducts, are subjectedto a third incubation period. During the third incubation period, thefollowing conditions are maintained: at a temperature of 36-37.2° C.(preferably 37° C.), in a medium of 7% rabbit serum, 5-10 days(preferably 8 days), 0-5% CO₂. The rabbit serum medium is periodically(eg. every 3-4 days) replaced. All spontaneously destroyed unwantedcells comprising exocrine cells, blood cells and elements of connectivetissue are removed.

As a result of the third incubation, the inventor obtained single-layercultures comprising progenic cells—precursors of beta-islet cells. It isimportant to note that these cells have an ability to grow in quantityon account of mitotic division. After the first and the secondincubation periods, a process of maturation of these cells can beobserved with the formation of insulin-producing beta-islet cells duringthe third incubation period.

The method described above maximizes the yield of beta-islet cells thatcan be generated from the rabbit pancreases. Using the methods developedby the inventors, the recovered beta-islet cells and progenic cells aremaximized.

The cell cultures obtained can be administered to diabetic patientswithout the use of immunosuppression agents.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, a “subject” is an individual and includes, but is notlimited to, a mammal (e.g., a human, horse, pig, rabbit, dog, sheep,goat, non-human primate, cow, cat, guinea pig, or rodent), a fish, abird, a reptile or an amphibian. The term does not denote a particularage or sex. Thus, adult and newborn subjects, as well as fetuses,whether male or female, are intended to be included. A “patient” is asubject afflicted with a disease or disorder, for example, diabetes. Theterm “patient” includes human and veterinary subjects. As used herein,the term “subject” can be used interchangeably with the term “patient.”

For use in the methods described herein, the compositions may beadministered by any means known to those skilled in the art, including,but not limited to, intraperitoneal, parenteral, intravenous,intramuscular, subcutaneous, or intrathecal. Thus the compositions maysuitably be formulated as an injectable formulation. Administration ofthe compositions to a subject in accordance with the invention appearsto exhibit beneficial effects in a dose-dependent manner. Thus, withinbroad limits, administration of larger quantities of the compositions isexpected to achieve increased beneficial biological effects thanadministration of a smaller amount. Moreover, efficacy is alsocontemplated at dosages below the level at which toxicity is seen.

By the terms “treat,” “treating” or “treatment of” (or grammaticallyequivalent terms) it is meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved and/or there is an inhibition or delay inthe progression of the condition and/or prevention or delay of the onsetof a disease or illness, for example, diabetes. The terms “treat,”“treating” or “treatment of” also means managing an autoimmune diseaseor disorder. Thus, the terms “treat,” “treating” or “treatment of” (orgrammatically equivalent terms) refer to both prophylactic andtherapeutic treatment regimes.

As used herein, a “sufficient amount” or “an amount sufficient to”achieve a particular result refers to a number of cells of the inventionthat is effective to produce a desired effect, which is optionally atherapeutic effect (i.e., by administration of a therapeuticallyeffective amount). For example, a “sufficient amount” or “an amountsufficient to” can be an amount that is effective to alter the severityof the subject's condition.

A “therapeutically effective” amount as used herein is an amount thatprovides some improvement or benefit to the subject. Alternativelystated, a “therapeutically effective” amount is an amount that providessome alleviation, mitigation and/or decrease in at least one clinicalsymptom. Clinical symptoms associated with the disorders that can betreated by the methods of the invention are well-known to those skilledin the art. Further, those skilled in the art will appreciate that thetherapeutic effects need not be complete or curative, as long as somebenefit is provided to the subject.

The culture method of the invention include the use of a selectedtemperature regimen of incubation for minced pancreatic micro fragmentsobtained from a rabbit and cultured in a serum free medium. Followingthe method of the invention, a pure preparation of viable and activebeta islet cells is obtained, which may be successfully transplanted toa diabetic patient without the use of any immune suppression.

The culture method of the invention provides favorable conditions forthe pancreatic beta cells and creates unfavorable conditions for ballastand immunogenic cells (so-called passenger cells) such as, for example,exocrine, endothelial, dendritic, and lymphocytes. The elimination ofall these passenger cells provides significant reduction of theimmunogenicity of the cultured islet cells. Contrarily to the commonbelief that long-term cultivation of cells required addition of a serumsupplement to the culture fluid, the inventor discovered that the use ofserum free media in the method of the invention favors the growth ofdesired beta islet cells and disfavors the growth of unwanted passengerscells and therefore provides viable and active beta islet cells in acommercially valuable amount, wherein such cell are substantially freeof unwanted passengers cells.

Without being committed to a particular theory, the inventors believethat shedding of surface antigens takes place in the cells prepared bythe methods of the invention, since there has been a remarkable lack ofimmune reaction despite the absence of any type of immunosuppressivetherapy neither before nor after clinical islet cellxenotransplantation. As highly refined culture of beta-cells prepared bythe method of the invention does not contain cells relating to donorvessels, so there is no rejection of xeno-transplanted beta-cells.

A synergistic combination of cells, solutions and conditions has beendiscovered by the inventors. This is accomplished by changing theenvironment, e.g., placement of the culture in O₂ incubator vs. 5% CO₂incubator, varying the temperature and time intervals in the incubatorover a period of 10-14 days. No genetic manipulations were used.

The culture technique and techniques of intra-muscular transplantationof islet cell culture will now be described in detail. Rabbits areharvested by submerging in 40-96% ethanol, preferably 70% ethanol untilthe death is registered, e.g., for up to 10 min, preferably 7-8 min. Insterile conditions, a pancreas is removed from the abdomen cavity of anewborn rabbit (the term “newborn” as used herein includes rabbits fromthe time of birth to 5 days old rabbits which were not exposed to breastmilk, preferably newborn to 12 h old rabbits) and immediately placed ina Petri dish with cold (e.g., 4-6° C.) Hank's salt solution orphysiological solution (NaCl) and antibiotics (e.g., penicillin 1000units/ml and streptomycin 10000 mg/ml). With the help of ophthalmictweezers, a capsule of pancreas is removed; vessels and excretory ductsare removed. Next, the pancreas is cut by an ophthalmic scissors intomicro fragments in the size of about 2-3 mm and then transferred to aspecial watch glass. Continue processing 18-20 pancreases in the abovedescribed manner. Next, pancreatic micro fragments are cut with theophthalmic (corneal) scissors into smaller pieces, i.e., mincedpancreatic micro fragments. The obtained minced pancreatic microfragments are washed out with cold Hank's solution and placed in aculture flask or a bottle (e.g., space of 75 cm², Corning-Costar)distributing them on a surface of the bottom of the flask. 5-7 minuteslater, during which there is an attachment of micro fragments toplastic, a serum free growth medium 199 (Sigma Aldrich) without a serumsupplement is poured into the flask (e.g., 10% of a flask volume). Theflask is then placed into an incubator with submission 0-5% CO₂ andpancreatic tissue is incubated at 36-37.5° C. (the first incubationtemperature), preferably 36.6-37° C. for 6-10 days, preferably 7-8 days(the first incubation period). Every 1-2 days, the flasks withcultivated cells were observed through the inverted microscope andspontaneously destroyed exocrine cells of pancreas, blood cells andelements of connective tissue were removed; the growth medium 199 wasreplaced with a fresh growth medium. As a result of the first incubationperiod, islet cell clusters formed at the bottom of the flask and atleast 78% of them (according to specific coloring) were pancreatic betacells.

For the final clearing of ballast cellular elements initiating theimmune response (e.g., leucocytes-passengers), culture flasks are placedin an incubator at temperature ranging from 22° C. to 29° C. (the secondincubation temperature) for 4-5 days (the second incubation period). Ithas been observed that at the second incubation temperature of 24° C.,the best results are achieved and the beta cell contain the least amountof passenger cells. After that the culture consisting only of isletcells 78-90% beta cells can be transplanted to a diabetic patient. Inaddition to islet cells, singular fibroblasts are found in the culturebut their share, usually not exceeds 1-5%. Cells of epithelial originare usually remaining cells in culture and make 5-17%, which fact theimmune-histo-chemical coloring (with monoclonal antibodies againstprotein CytoKeratin 18) confirms; but they are not beta-cells, as thesecellular structures are not revealing presence of insulin.

One dose of islet cell culture preferably containing 1,400,000-1,600,000cells in a sterile suspension in Hank's salt solution (10-15 ml volume,not cranial, all cells obtained) can be placed to a plastic tube markedin an appropriate way.

Islet cell culture made in accordance with the method of the invention(also referred herein as Beta Cells of Philadelphia Medical ScientificCenter or BCPMSC) has following cell composition:General cell count: 1,500,000+100,000Beta cells 82+8% (at least 50%)Other islet cells +30 9+2%

Fibroblasts +30 2+1%

Progenitor (stem) islet cells +30 7+3%Cultivation is performed in serum-free growth medium 199.Period of storage of BCPMSC:

-   -   at 4-10° C. for 72 hours    -   at 11-24° C. for 48 hours    -   at 30-37° C. for 12 hours.        The dose of islet cell cultures was prepared from 80 1-2 day old        newborn rabbit's pancreases.        Term of cultivation—14-21 days.        Cell viability—82% or more is preferred.        At control incubation of the cultured islet cells, the insulin        basal production has made 8400±1200 μU/ml/h,        stimulated—16500±2700 μU/ml/h        The culture method of the invention excluded contamination of        the culture by microorganisms (e.g., bacteria, fungi, mycoplasma        and viruses).

Each dose will be accompanied with a certificate describing preparationhaving all necessary information on BCPMSC. Criteria for the isolationor elimination of diseased animals; as stated, at the first sign of anydisease an animal is quarantined and is never returned back to thecolony from quarantine.

The obtained culture can be transplanted to a recipient, e.g., aDiabetes Mellitus (DM) patient. The transplant (xenograft) or aninjection can be administered by various ways, for example, by injectioninto the liver (i.e. directly into the hepatic parenchyma or through theportal vein), into pulp of the spleen, into the splenic artery, inspecially performed large omentum pocket with use of laparoscopytechnique, and intramuscularly. The preferred method of introduction ofculture is into the rectus abdominis muscle of the recipient.

An objective of the invention is to harvest islet cells from rabbitpancreases, which do not contain any immunogenicity and therefore do notrequire immunosuppression after an allogeneic or xenogeneictransplantation to patients with Diabetes Mellitus. The novelty of thistherapy is that all existing therapies that obtain islets from a donorpancreas, are immunogenic (contain vascular endothelial, as well asblood cells etc.) and after transplantation of such islets,immunosuppression is necessary.

Recently, we made attempts to apply such methodological approaches,which would allow us to achieve the similar therapeutic (anti-diabetic)effect while utilizing a lesser amount of donor pancreases.

The inventors noticed that in the culture obtained according to themethod described above, the viability of some singular beta-islet cellsor small beta-islet cell clusters which account for about one half ofthe total mass of the culture is up to 60-70% (based on vital coloringby Trypan Blue) after a relatively long (2-3 weeks) incubation period invitro. By the end of 6 weeks of cultivation the viability of the cellsdoes not exceed 40%. However, at the same time, in larger clusters theviability of beta cells is no less than 80% even after 8-10 weeks ofcultivation.

As a result, the inventors developed a method of maximizing the yield ofbeta-islet cells from rabbit pancreases. The method is described indetail below.

Harvesting the culture of beta-islet cells from the pancreases ofnewborn rabbits comprising only beta-islet cells and free of vascularendothelia, leukocytes and other cellular products which can initiaterejection of the transplanted cultures by the immune system of therecipient. The procedure of preparing micro fragments from the new bornrabbit pancreases is described above. For example, harvesting thepancreases of newborn rabbits and placing pancreases in a salt solutioncomprising an antibiotic at a temperature of 4-10° C.; removing vesselsand excretory ducts from the harvested pancreases; obtaining mincedpancreatic micro fragments from said pancreases.

The micro fragments are transferred into a receptacle container, such asa medical or laboratory roller bottle, that is adapted for a rotatingdevice, with a serum-free medium. An example of the serum-free media isMedium 199. The rotating device is housed in an incubator.

The minced pancreatic micro fragments in the serum-free medium areincubated in the rotating device at a constant speed of 2-10 revolutionsper minute (preferably 2 to 6 RPM) at a first incubation temperature 36°C. to 37.2° C. (preferably 37° C.) for 5 to 12 days (preferably 8-10days) at 0% to 5% CO₂ for a first incubation period. During theincubation period, the serum free medium is periodically (eg. every 3-4days) replaced, and the spontaneously destroyed unwanted cellscomprising exocrine cells and blood cells and elements of connectivetissue are removed.

After a few days during the first incubation, free floating beta-isletcells associate into more dense neo-islet cells that are sphericalstructures with a diameter of 80 to 150 microns. Such island-likeneo-islet cells comprise approximately 78-90% beta-islet cells. Theneo-islet cells so obtained can be used directly in transplantation todiabetes patient without immunosuppressive therapy as the neo-beta isletcells comprise only non-immunogenic beta-islet cells.

The neo-beta islet cells can be administered in a medically therapeuticquantity either hypodermically, intramuscularly, intra-abdominally viaabdominal musculature (Rectus or other abdominal muscle), into amesenteric vessel of the small intestines, into the Greater Omentum,directly into the liver via the hepatic vascular system, arterial systemor intraparanchymal injection or into the spleen through the splenicartery.

Also formed during the first incubation period are clusters ofbeta-islet cells that are up to 1 mm in diameter. The beta-islet cellclusters are harvested and further incubated for a second incubationperiod. During the second incubation period, the following conditionsare maintained: a temperature of 18-28° C. (preferably 24° C.), aduration of 4-10 days (preferably 6-8 days), a serum-free medium (suchas medium 199), and 0-5% CO₂. During the second incubation period, theserum free medium is periodically (eg. every 3-4 days) replaced, allspontaneously destroyed unwanted cells comprising exocrine cells, bloodcells and elements of connective tissue are removed, and beta-islet cellclusters remained in at least 80%.

Like the neo-islets, the beta-islet cell clusters obtained after thesecond incubation can be administered in a medically therapeuticquantity either hypodermically, intramuscularly, intra-abdominally viaabdominal musculature (Rectus or other abdominal muscle), into amesenteric vessel of the small intestines, into the Greater Ommentum,directly into the liver via the hepatic vascular system, arterial systemor intraparanchymal injection or into the spleen through the splenicartery.

It is known that precursors of mature beta-cells are located inepithelium of fine excretory ducts of pancreases. These cells can beattributed to regional stem cells or progenic cells. As a source of suchcells we used tissues of micro fragments of pancreases of newbornrabbits that remained after the first incubation period described above.During these days, in general, death and elimination of exocrine cellsoccurs, as well as migration of beta cells. Remains of pancreaticfragments, comprising in general of fine excretory ducts, are subjectedto a third incubation period. During the third incubation period, thefollowing conditions are maintained: at a temperature of 36-37.2° C.(preferably 37° C.), in a medium of 7% rabbit serum, 5-10 days(preferably 8 days), 0-5% CO₂. The rabbit serum medium is periodically(eg. every 3-4 days) replaced. All spontaneously destroyed unwantedcells comprising exocrine cells, blood cells and elements of connectivetissue are removed.

As a result of the third incubation, the inventor obtained single-layercultures comprising progenic cells—precursors of beta-islet cells. It isimportant to note that these cells have an ability to grow in quantityon account of mitotic division. After the first and the secondincubation periods, a process of maturation of these cells can beobserved with the formation of insulin-producing beta-islet cells duringthe third incubation period.

The method described above maximizes the yield of beta-islet cells thatcan be generated from the rabbit pancreases. The first incubation givesrise to: 1) pure neo-islets which can be directly used in thetransplant; 2) clusters of beta-islet cells; and 3) residual tissues ofmicro fragments of pancreases of newborn rabbits. The beta-islet cellclusters are subject to a second incubation from which highly pure andhighly viable cells are obtained. The tissues of micro fragments ofpancreases are incubated to produce a composition comprising progeniccells (precursors of beta-islet cells) and insulin-producing beta-isletcells. Using the methods developed by the inventors, the recoveredbeta-islet cells and progenic cells are maximized.

As quantitative analyses and morphometric studies demonstrate, totalcell mass of the culture after applying roller cultivation can beincreased by approximately two-fold compared to the original methoddescribed in U.S. application Ser. No. 12/094,721 (published as U.S.2008/0267925). Hence, utilization of these methods of preserving andincreasing the amount of beta cells produced from rabbit pancreasesallows a decrease in the needed animal-donors by from one half totwo-thirds.

The neo-islets, beta-islet cell clusters, and the composition comprisingprogenic cells (precursors of beta-islet cells) and insulin-producingbeta-islet cells obtained can be pooled together before administration.

Transplantation Technique

With the injection needle, preferably not less than 7 cm in length andnot less than 1 mm in diameter, the islet cell suspension is collectedin a syringe and injected into, for example, the musculus abdominisrectus after local anesthesia. Preferably, the site of an injection isto be closed by sterile bandage. The invention provides a method whereinthe administration is, for example, intraperitoneal, parenteral,intravenous, intramuscular, subcutaneous, or intrathecal. The inventionprovides a method wherein the administration is, for example, into liverthrough portal vein, directly into parenchyma of the liver, or intospleen.

Proof of Reduced Immunogenicity of the Islet Cell Cultures

Evidence in vitro: various histological experiments including light andelectronic microscopy; immunohistochemistry, shown above, demonstratedthat in the pure culture there are no so-called leucocytes-passengers(lymphocytes, macrophages and etc.) capable of initiating immunereaction after the procedure (9-12 month). Evidence in vivo:xenotransplantation of the islet cell culture into rats with anexperimental diabetes leads to remission of the diabetic status for atleast 30 days.

For the purpose of studying of immunogenecity of cells containing in theculture obtained through the above-described method from the pancreas ofnewly-born rabbits, there were experiments conducted to determinefixation of immune-globulins of human blood serum on them. Cells,incubated with various human blood serums were stained by monoclonalantibodies against human immune-globulins and analyzed on the flowcytometer. It appeared that cells containing in the culture are capableof fixating on their surface immunoglobulins M, but there were noimmunoglobulin G fixation discovered.

A proper preparation of the diabetes patient for islet cell culturetransplantation is recommended. Before the transplant treatment, adiabetic patient has to achieve as good glycemic state as possible byintensive insulin therapy. Patients should have no vaccinations and noserum therapy for 4 weeks prior to cell transplantation. Clinicalresearch should include consultations of ophthalmologists,nephrologists, neurologist, vascular surgeon, dermatologist,diabetologist, and consultations of other specialists regardingsecondary diabetic complications.

A patient can be prepared for islet cell xenotransplanation in thefollowing manner:

-   -   1. Elimination of ketoacidosis, frequent hypoglycemia or        hyper-osmolarity by hospitalization, so that the patient's        clinical condition will be compensated as possible;    -   2. Maximal compensation of diabetic status, stabilization of        blood sugar within normal or near normal levels by adequate        insulin therapy under tight self-control of glycemia;    -   3. Avoidance of vaccinations or serum therapy for 4 weeks prior        to cell transplantation.        The following parameters will be followed in patients before and        after the islet cell xenotransplanation, with the frequency as        follows:

General:

-   -   1. Level of HBA-1c (clycosylated hemoglobin) every 3 months;    -   2. Level of C-peptide (basal and stimulated) every 3 months;    -   3. Detection of auto-antibodies; anti-GAD, anti-insulin,        anti-ICA;    -   4. Serum cholesterol and triglycerides every 3 months;    -   5. Home blood glucose self-monitoring (with diary) several times        a day;    -   6. Correction of insulin requirements (with diary).

Special:

Diabetic Retinopathy:

-   -   1. Standard retinal fundal field evaluation by sterofundoscopic        photography and fluorescent technique every 3 months;    -   2. Visual acuity every 3 months.

Diabetic Nephropathy:

-   -   1. Proteinuria/24 hr once a month;    -   2. Microalbuminuria every 3 months;    -   3. Serum creatinine every 3 months;    -   4. Creatinine clearance very 3 (60 months;    -   5. Blood pressure once a week.

Diabetic Neuropathy:

-   -   1. EMG every 3 months;    -   2. Nerve Conduction studies of tibial nerve; sural nerve; median        nerve—every 3 (6) months    -   3. Pain analog scale once a month;    -   4. Orthostatic changes—once a month;    -   5. EKG—r-r variation every 3 months.

Diabetic Vasculopathy:

-   -   1. Doppler ultrasound every 3 (6) months;    -   2. Doppler probe;    -   3. Doppler Blood pressure ankle/arm;    -   4. Doppler segmental blood pressure;    -   5. Plethsmograph waveform change.

As biochemical and morphological research has demonstrated, cultures ofpancreatic insular cells produced by the stated methods possess highsecretory activity and sharply reduced immunogenic (immunologicalpotency). A certificate of quality is preferably attached to eachportion of cells designed for cell-therapy.

Indications and Contraindications for Xenotransplantation of IsletCultures

Exemplary indications: (a) labile course of insulin dependent DiabetesMellitus (IDDM) with inclination towards hypoglycemic status and/orketoacidosis, (b) inability of reaching satisfactory compensation ofIDDM by usual methods; insulin-resistance, (c) secondary complicationsof diabetes mellitus in patients with IDDM and NIDDM (neuropathy,nephropathy, retinopathy, angiopathy of lower extremities, etc.),excluding terminal studies, and (d) IDDM (insulin dependent) and NIDDM(non-insulin-dependent) without detection of secondary complications—forthe purpose of prophylaxis.

B. Contra-indications: Acute infections & inflammatory diseases, orexacerbation of chronic diseases; Oncological Diseases.

Supervision of Patients after the Transplantation of Islet Cell Culture

Within the first year after transplantation, the above-stated inspectionafter 3, 6, 9 and 12 months is recommended.

The majority of recipients in 1-3 months after xenotransplantationdemonstrate the following changes:

-   -   1. Current/prior to XT—labile 1 type diabetes is stabilized;    -   2. The bent of patients to ketosis disappears;    -   3. Parameters of carbohydrate exchange (it is reduced daily        average glycemia—in 1-2 months, the decrease of glycosylated        hemoglobin content—3 months) improve;    -   4. Decreases (for 20-30%) of requirement for exogenous insulin;    -   5. Parameters of lipid metabolism improve:    -   6. At recipients with absence of residual secretion of insulin        by own beta cells (it is C-peptide not determined) appears the        production of patient's insulin (C-peptide).    -   7. At patients with sensory motor neuropathy, pain and        paresthesia disappear; parameters of sensitivity and        conductivity of fibres of peripheral nerve improve. At patients        with autonomic neuropathy, who struggle with glycemic control,        postural hypotension, gastroparesis, and enteropathy (diarrhea),        parameters of pulse and blood pressure are normalized, the        functions of stomach and gut normalized too.    -   8. At 1 type diabetes patients with a stage expressed diabetic        nephropathy (classification on C. Morgensen) decreases and        disappears proteinuria, high blood pressure is reduced and        normalized. At patients with a 3^(rd) stage of diabetic        nephropathy, microalbuminuria decreases or becomes normal (less        than 30 mg/day).    -   9. At patients with nonproliferated and preproliferated states        of diabetic retinopathy, the clinical picture of a eye bottom is        stabilized, the significant part of recipients have its        improvement: haemorrhages resolve, the hypostasis of a retina        decrease.

Stabilization of a diabetes mellitus course and the lowering of theexogenous insulin requirement result from the adequate functioning oftransplanted islet cells and a partial recovery or increased function ofislet cells of recipient's pancreas.

It is believed that the curative effect of transplanted cell cultures onthe late diabetic complications is apparently explained by therestoration or strengthening of secretion by both transplanted and apatient's own beta cells of C-peptide, which is produced together withinsulin and has a marked angioprotective effect.

According to hypothesis the development of microangiopathy, which is thebasis of all diabetic complications, is due to the lack of C-peptide andsome other hormone-like substances produced by the beta cells, which areabsent at overwhelming majority of 1-type diabetes patients.

C-Peptide injection in patients with complicated 1-type diabetesmellitus results in the regress of secondary diabetic complications,such as nephropathy, retinopathy and neuropathy.

Transplanted rabbit's beta cells produce normal C-peptide, which has aphysiological influence on late diabetic complications.

Table 1 demonstrates improved glycemic control and drop in insulinrequirement following ICCXT (well-documented 112 cases).

TABLE 1 Before 3 Mo 6 Mo 9 Mo 12 Mo Index XT After After After AfterAverage Daily 198 + 41 158 + 52  129 + 23  133 + 30 142 + 34 Glycemia,mg/dl HbAlc, % 10.1 + 2.1 9.1 + 1.2 7.7 + 1.9  6.9 + 1.1  7.5 + 1.9Insulin Dose,  56 + 11 38 + 15 25 + 12 32 + 9 40 + 8 IU

Table 2 demonstrates insulin requirement (IU/day) and microalbuminuria(mg/day) level after repeated ICC XT in patient Ts.S (33 years old, 18years of 1-type DM duration)

TABLE 2 Before 3 Mo 6 Mo 9 Mo 12 Mo XT Index XT After After After After1^(st) Insulin dose 64 33 38 40 48 Microalbuminuria 936 680 348 330 4962^(nd) Insulin dose 52 38 24 26 38 Microalbuminuria 660 377 189 199 1673^(rd) Insulin dose 42 38 25 32 40 Microalbuminuria 330 145 99 112 884th Insulin dose 40 34 20 16 16 Microalbuminuria 66 45 34 40 39

The main advantage of transplantation of islet cells in comparison tothe usual therapy of Diabetes Mellitus of the 1-type, are as follows:

Due to regularly performed transplantation, all recipients show decreasein progression of diabetic angiopathy, and reversion of initial stagesof late diabetic complications (retinopathy, nephropathy, neuropathy,and angiopathy), which is impossible to achieve with the help of usualtherapy (injection of insulin upon self-control of glycemia, andtraditional treatment of angiopathy).

This happens mostly because the transplanted beta-cells after thetransplant taking (acceptance) start to produce in a recipient's body anangio-protective matter C-peptide, which a patient was deprived due tothe collapse of his own beta-cells (due to auto-immune assault).

Ability of cultures of pancreatic insular cells, received through theoriginal method from pancreas of newborn rabbits, to survive andfunction in in-vivo environment, ahs been demonstrated by us inexperiments of xenotransplantation of such cultures to animals withexperimental Diabetes Mellitus.

Vistar Line male rats of body mass of 180-220 gm, regularly fed, wereused as experimental animals.

Experimental Diabetes Mellitus was provoked by sub-dermal application ofAlloxan (dosage 200 ml for 1 kg of body weight) or by sub-dermalapplication of Streptozotocini (dosage 60 ml/kg).

During experiments and control probes we used only rats with Alloxan orStreptozotocin-induced Diabetes, those whose level of hypoglycemia onempty stomach was 20 mmol/l and higher. Earlier conducted testsindicated that such animals did not have spontaneous reversion ofexperimental Diabetes Mellitus.

After transplantation of pancreatic islet cell (P.I.C.) cultures, 88 outof 104 rats with stable or sever Alloxan-induced Diabetes Mellitus(almost 85%) displayed firm remission of diabetic status up-to the endof experimental term (20 weeks). Firm decrease of blood sugar levels ofalmost-up-to-normal levels was registered in blood ofanimals-recipients. At the same time, characteristics clinical symptomsof diabetes were also vanishing (such as weight loss, polydipsia,polyuria). Anti-Diabetic effect of xenotransplantation was clearlydemonstrated both in cases of application of cultures into liver(through portal vein or directly into liver's parenchyma) and also intospleen (cultures were brought in intra-pulp), and also through theabdominal muscles. Even after 8 weeks after xenotransplantation, P.I.C.with preserved structure and with signs of secretory activity wasdetected in places of implantation in rats with remission ofexperimental Diabetes.

During special series of experiments the role of preliminary cultivationof P.I.C. in vitro was demonstrated clearly in survival of cells inorganisms of xenogeny recipient. For that purpose, we performedcomparative analysis of results of xenotransplantation of cultures ofP.I.C. of pancreas of human fetuses and xeno-transplantation ofnon-cultivated fetal Island tissue to rats with experimental DiabetesMellitus. It was detected that sugar-reducing effect is more expressedand long-lasting in cases of transplantation of pre-cultivated P.I.C. incomparison with transplantation of non-cultivated tissue of Pancreas,which results only in short-lived remission of Diabetic status. So, theimmune-modulating result of cultivating in vitro was experimentallyproven to significantly increase the term of survivability in anorganism of alien recipient.

Pancreases of 18 rats-recipients, on whom successful xenotransplantationof cultures of Pancreas of newborn rabbits had been performed, weresubjected to histological exam in 8 weeks after transplantation. Forthat purpose a fragment of pancreas was fixated in Buena solution andwas drown in paraffin. Slices (5-7 mkm thick) were colored byhematoxilin and eosin, and also by Aldegid-Fuxin for revealing ofβ-cells. At the same time, pancreases of 6 control animals who haduntreated alloxan-induced Diabetes as well as pancreases of 6 healthyrats (no Alloxan applied) were closely examined.

While examining pancreases of healthy intact rats, some 45-76% ofβ-cells, as expected, were found in ‘Langerhans” islands. Rats withuntreated Alloxan-induced Diabetes had sharply decreased amount ofβ-cells in Islands—in average 8.3+−1.1%.

Significantly higher amount of β-cells in Islands was discovered inrats-recipients. In animals, who had been subjected toxenotransplantation of P.I.C. cultures, their own Pancreases displayedtypical β-cells and its share among “island” cells was from 10 to 55%(some from 7 to 21%) (average 23.5+8.8).

With regards to these experiments, we may assume that anti-diabeticeffect of xeno-transplantation of OK cultures on developments ofexperimental diabetes in rats is occurring 2 general ways: a)Functioning of transplanted β-cells, confirmed in addition to expressedsugar-reducing effect, also by revealing groups of transplanted P.I.C.in the pulp of spleen of animals-recipients: b) Stimulating effect oftransplantation of P.I.C. cultures on the Island apparatuses of pancreasof rats-recipients, which possibility is confirmed by data histologicalexams revealing existence of significantly frequent of Islands withnormal β-cells and bigger share of them in Islands of pancreas ofrats-recipients than of rats with untreated Alloxan-induced Diabetes.

Successful experimental research became grounds for performing clinicaltransplantation of cultures of pancreas of newborn rabbits toDiabetes-type-1 patients.

Clinical Transplantation of Cultures of P.I.C. Produced Out ofPancreases of Newborn Rabbits.

Total of 112 patients with Type-1 Diabetes Mellitus (IDDM) were underwell-documented dynamic supervision.

Of total of 112 patients there were 58 men, and 54 women. Patients' ageat the moment of transplantation varied from 16 to 53—average 35 yearsold.

It is known that severity of manifestation of secondary diabeticcomplications depends significantly on duration of the disease IDDM.Supposedly, destruction of own β-cells of the patient as a result ofautoimmune process approximately happens on the 5^(th) year aftermanifestation of the disease. Secondary diabetic complications manifestitself usually in patients with duration of the disease of more than 10years. Because of that, all IDDM patients were divided in 3 groups inreference to duration of the disease: a) 1 to 5 years—16 people; b) 6 to10 years—43 people c) more than 10 years—53 patients. All patients hadbeen examined with the aim to determine character of development of IDDMand establishing presence of diabetic complications.

Usually cultures received through the above-described methods out of50-60 pancreases of 1-2 day newborn rabbits were used fortransplantation for one patient. Suspension was usually delivered intotransverse abdominal muscle under local anesthesia. No immunesuppression was used.

The most important fact confirming an anti-diabetic effect in isletcells transplantation just because of functioning of transplantedbeta-cells, is the fact of discovering them at the place ofadministering the transplantation. If after intra-peritonealtransplantation it is almost unreal to locate applied cells in theabdominal cavity, then after administering into the spleen it ispossible, although very difficult.

A microphotograph of colored histological section through the spleen'spulp was obtained which clearly demonstrates a transplant represented bya group of epithelial cells in the center of the photo. Assurance thatthese cells are indeed a transplant is based, partially on the groundsthat epithelial structures are absent in splenic tissue.

Accordingly, presence of epithelial cells in lineal pulp bears evidenceto “incomer from outside”, in this case—a transplant.

Based on the results of the scientific-experimental research, theseveral basic conclusions can be made:

-   1. Streptozotocin (Stz) makes general destructing effect on    beta-cells of islets of pancreas, but at the same time, directly or    indirectly leads to a loss of other islet cells.-   2. It appears that a regeneration process in affected Langerhans    islets happen mainly on account of recovery of beta-cells pool.-   3. Islet cells cultures produced from pancreases of newborn rabbits    through the original method consist generally of beta-cells cleared    of ballast cellular elements and have a very high insulin-producing    activity.-   4. An intra-peritoneal, as well as intra-splenetic    xenotransplantation of islet cells cultures to rats with    experimental Streptozotocin induced Diabetes Mellitus, provides, in    majority of cases, a stable remission of diabetic status for a    duration of at least 8 week period.-   5. Post-transplantation sugar-decreasing effect is secured as by    functioning of transplanted beta-cells, also by insulin-producing    activities in, to some degree, restored pool of beta-cells in islets    of pancreases of rats-recipients. This is confirmed by findings of    measuring concentrations of exogenous (rabbit's) and own (rat's)    insulin in the blood of experimental animals.-   6. Histological examinations of pancreases of experimental rats    confirmed the expressed stimulation of regenerative processes in    islets of rats with Streptozotocinal Diabetes Mellitus after    administering them xenotransplantation of islet cells cultures.    It is possible that regeneration of beta-cells happens not only in    the margins of localization of Langerhans islets but also in some    structures outside of islet pancreatic tissue.

The invention will be illustrated in more detail with reference to thefollowing Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1

At the initial stage of scientific-research work the inventors used ratsof Vistar line of body mass 220-250 grams received from a specialnursery of lab animals as experimental animals. In order to eliminatehormonal cyclic influences on alteration of parameters of carbohydratemetabolism, we decided to carry on experiments on mature pubescentmales.

In order to obtain objective results in conducting antidiabetictreatment in lab rates there was designed a model of a stable diabetesmellitus.

Disease in animals was provoked by introduction of fractionalintraperitoneal administration of derivable ex-tempore solution ofStreptozotocin—total dose of 80 mg per 1 kg of body weight. Total of 90animals were subjected to effect of streptozotocin. Later majority ofanimals developed characteristic signs of diabetic status: thirst,polyuria (excessive blood glucose), polyfagia (excessive food intake),hair fallout, slowing of body mass gain or its decline. At that, anexpressed hyperglycemia was registered in 58 rats, i.e. other 22retained normal glycemia or concentration of glucose rouseinsignificantly. By the 4-week term after inducing of diabetes 32animals with more stable level of hyperglycemia were picked over.Stability was confirmed by the fact that during almost a month-longobservation, the concentration of blood sugar did not go lower than 16mmol/l.

Choice of initial glycemia of such range is not on a chance basis as itis explained by the following. As previous experiments demonstrated, inglycemia of level of 16 mmol and higher, as registered in not less thanin 4 weeks after administering Streptozotocinal, such rats do notdemonstrate a spontaneous remission in the future as well as a reversionof a diabetic status. At the same time even a very high glycemia (stillunder 30 mmol/l) lets the majority of experimental animals with diabetesto survive for long periods (2 and more months), which allows conductingrather continuous experiments without any serious concerns for anuntimely death of such animals. These assumptions were confirmed byobservation over the control (no treatment) group of diabetic rats.Below are findings on an effect of intra-peritoneal xenotransplantationof cultures of islet cells of newborn rabbits on the course ofStreptozotocinal Diabetes in 8 rats with consistent StreptozotocinalDiabetes Mellitus.

Each of animals-recipients, on the background of Hexenalintra-peritoneal narcosis, was administered 700,000-800,000 islet cellsof pancreases of newborn rabbits by administration it through puncturein abdominal wall by a big diameter needle.

Below are findings regarding change in severity of diabetic status ineach of 8 experimental animals of this group.

The animals were divided in 3 groups of 8 rats each.

-   1^(st) group: 8 rats with hyperglycemia, to whom xenotransplantation    of cultures of    -   islet cells into abdominal cavity was administered;-   2^(nd) group: 8 rats with hyperglycemia, to whom xenotransplantation    of cultures of islet cells was administered into spleen pulp;-   3^(rd) group: 8 rats with hyperglycemia, who were not subjected to    any treatment (control).

TABLE 3 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intraperitoneal transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #1. 4Prior weeks Prior to after to Weeks Indexes Stz Stz Tx 1 2 3 4 5 6 7 8Glycemia 6.7 20.0 24.1 12.2 12.0 12.4 12.5 12.2 11.9 14.8 14.9 mM/l Body220 175 170 180 200 230 250 290 330 340 Mass, g

TABLE 4 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intraperitoneal transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #2. 4Prior weeks Prior to after to Weeks Index Stz Stz Tx 1 2 3 4 5 6 7 8Glycemia 6.4 18.7 16.6 13.0 6.8 6.7 7.4 6.4 5.5 7.0 6.0 mM/l Body 200180 170 180 210 230 240 250 270 290 320 Mass, g

TABLE 5 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intraperitoneal transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #3. 4Prior weeks Prior to after to Weeks Index Stz Stz Tx 1 2 3 4 5 6 7 8Glycemia 5.4 26.8 29.0 16.1 22.2 16.5 12.5 14.1 17.7 16.5 15.9 mM/l Body200 180 160 160 170 200 210 220 240 250 290 Mass, g

TABLE 6 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intraperitoneal transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #4. 4Prior weeks Prior to after to Weeks Index Stz Stz Tx 1 2 3 4 5 6 7 8Glycemia 6.0 19.7 21.2 12.2 13.0 14.1 9.2 7.9 9.0 9.2 8.3 mM/l Body 240210 200 190 210 250 260 270 310 320 350 Mass, g

TABLE 7 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intraperitoneal transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #5. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 4.9 18.9 17.9 15.5 15.9 14.0 15.9 15.0 15.2 16.8 14.6 mM/l Body200 180 170 180 200 220 240 250 270 280 300 Mass, g

TABLE 8 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intraperitoneal transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #6. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 5.9 22.3 21.9 14.2 11.1 7.8 7.0 8.4 8.1 7.8 7.4 mM/l Body 240210 210 200 240 240 260 290 320 350 360 Mass, g

TABLE 9 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intraperitoneal transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #7. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 5.9 18.0 17.1 11.5 7.9 8.1 6.4 5.4 6.1 5.1 6.4 mM/l Body 200180 170 180 200 220 240 250 270 280 300 Mass, g

TABLE 10 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intraperitoneal transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #8. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 6.3 24.0 25.6 17.7 18.8 20.0 21.8 18.6 17.8 18.8 20.9 mM/l Body250 190 170 190 200 220 250 260 270 290 300 Mass, g

Example 2

Eight (8) rats with stable Streptozotocinal Diabetes Mellitus weresubjected to intra-splenetic xenotransplantation of islet cell cultures.Administration of cultures of islet cells of pancreases of newbornrabbits was conducted on the background of Hexenal narcosis. Afteropening of an abdominal wall and bringing a spleen into an operativewound, a cellular suspension was introduced directly into the organ'spulp through the syringe of 1.2 mm in diameter. Place of injection waspressed with gauze tampon, by doing such slowing the parenchymalbleeding and sealing it by drops of special medical glue. Abdominal wallwound was stitched up layer by layer. Below are general results of suchtransplantations, exactly dynamics of indexes of glycemia and body massof animals-recipients.

TABLE 11 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intra-splenetic transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #17. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 5.3 16.7 17.1 16.8 12.2 10.2 10.0 11.2 9.9 8.1 6.6 mM/l Body220 200 200 190 200 240 250 280 320 340 360 Mass, g

TABLE 12 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intra-splenetic transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #18. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 6.6 24.0 22.1 19.5 18.0 12.6 7.4 6.5 5.7 7.0 10.2 mM/l Body 220180 170 170 180 200 240 290 320 330 360 Mass, g

TABLE 13 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intra-splenetic transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #19. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 5.3 21.2 21.2 17.6 14.4 10.5 11.2 11.4 9.1 10.5 7.4 mM/l Body250 220 210 190 200 210 250 270 300 310 350 Mass, g

TABLE 14 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intra-splenetic transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #20. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 6.7 24.5 26.9 14.2 12.8 13.0 8.8 8.4 9.0 7.4 9.8 mM/l Body 210170 170 180 180 200 220 240 270 290 330 Mass, g

TABLE 15 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intra-splenetic transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #21. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 6.0 22.0 21.9 15.5 15.9 14.0 13.5 15.1 13.8 17.0 15.5 mM/l Body250 180 170 170 180 200 220 230 230 240 270 Mass, g

TABLE 16 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intra-splenetic transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #22. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 7.1 31.4 28.8 24.2 25.9 22.7 17.0 17.9 18.1 16.7 15.8 mM/l Body220 170 170 160 160 180 200 220 230 250 270 Mass, g

TABLE 17 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intra-splenetic transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #23. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 5.6 16.0 15.5 11.5 9.7 6.6 7.5 5.6 6.6 5.9 5.7 mM/l Body 240260 270 290 300 320 350 370 370 400 410 Mass, g

TABLE 18 Change of glycemia and body mass after application ofStreptozotocin (Stz) and subsequent intra-splenetic transplantation (Tx)of islet cells cultures of pancreases of newborn rabbits in rat #24. 4Prior weeks Prior Weeks Index to Stz after Stz to Tx 1 2 3 4 5 6 7 8Glycemia 6.4 21.0 22.6 17.1 15.8 14.2 21.8 18.6 17.8 19.1 19.7 mM/l Body250 230 220 210 200 220 230 240 270 280 360 Mass, g

These data were later confirmed by observations over the control(diabetes without treatment) group of diabetic rats.

Example 3

26 animals were divided into 3 groups of 8 rats each: First group—8 ratswith hyperglycemia subjected to xenotransplantation of islet cellsculture through intraperitoneal administration. Second group—8 rats withhyperglycemia, which were subjected to xenotransplantation throughintra-splenetic administration. Third group—10 rats with hyperglycemia,which were not treated at all (control). Greater number of such animalsexplained by projection of their possible death in the course of anexperiment.

Below is data about an effect of intraperitoneal xenotransplantation ofislet cells culture of newborn rabbits on the course of Streptozotocinaldiabetes in 8 rats with expressed Streptozotocinal diabetes mellitus.

Each animal—recipient, on the background of Hexenal intraperitonealnarcosis, was introduced 700,000-800,000 of islet pancreas cells ofnewborn rabbits through puncture of peritoneum and release intoabdominal cavity.

Below are findings on alterations of severity of diabetic status in eachof the 8 rats of this group. Unlike rats with stable Streptozotocinaldiabetes that were administered either intraperitoneal or intra-splenictransplantation of islet cells cultures of newborn rabbits, rats withthe same severity of diabetic status that were not subjected to anytreatment (control), level of glycemia remained stable high during thewhole term of this experiment. In addition, no one rat-recipient died,but in a control group, on the background of a severe diabetic status 2animals died, which makes it a ⅕ of animals of the group.

Below, are findings on alterations of glycemia level and body mass inrats with untreated experimental diabetes mellitus.

TABLE 19 Change of glycemia and body mass after application ofStreptozotocin in rat #9 not subjected to transplantation (control).Prior 4 weeks Weeks Index to Stz after Stz 1 2 3 4 5 6 7 8 Glycemia 6.425.8 23.9 23.4 26.6 24.7 23.3 24.4 22.6 24.3 mM/l Body 250 230 210 200200 180 170 170 180 180 Mass, g

TABLE 20 Change of glycemia and body mass after administeringStreptozotocin in rat #10 not subjected to transplantation: Prior 4weeks Weeks Index to Stz after Stz 1 2 3 4 5 6 7 8 Glycemia 7.1 28.727.9 >33 29.9 28.4 >33 31.2 e.l.* mM/l Body 200 180 170 160 160 150 150130 Mass, g *e.l.—exitus letalis

TABLE 21 Change in Glycemia and body mass after administeringStreptozotocin to rat #11 not subjected to transplantation (control)Prior 4 weeks Weeks Index to Stz after Stz 1 2 3 4 5 6 7 8 Glycemia 6.426.8 24.9 23.4 26.6 25.7 27.3 24.9 25.5 23.7 mM/l Body 220 220 210 200190 180 170 180 180 180 Mass, g

TABLE 22 Change in Glycemia and body mass after administeringStreptozotocin to rat #12 not subjected to transplantation (control)Prior 4 weeks Weeks Index to Stz after Stz 1 2 3 4 5 6 7 8 Glycemia 6.422.8 21.3 23.4 27.6 24.1 25.3 24.7 22.5 23.7 mM/l Body Mass, g 220 200210 210 190 180 190 200 210 210

TABLE 23 Change in Glycemia and body mass after administeringStreptozotocin to rat #13 not subjected to transplantation (control)Prior 4 weeks Weeks Index to Stz after Stz 1 2 3 4 5 6 7 8 Glycemia 6.421.8 23.0 23.7 24.6 23.6 28.3 24.8 25.0 23.8 mM/l Body Mass, g 220 240230 220 210 200 190 180 190 200

TABLE 24 Change in Glycemia and body mass after administeringStreptozotocin to rat #14 not subjected to transplantation (control)Prior 4 weeks Weeks Index to Stz after Stz 1 2 3 4 5 6 7 8 Glycemia 4.926.6 24.5 25.6 27.7 23.6 25.9 25.0 27.2 28.8 mM/l Body Mass, g 200 200210 200 180 170 180 170 160 160

TABLE 25 Change in Glycemia and body mass after administeringStreptozotocin to rat #15 not subjected to transplantation (control)Prior 4 weeks Weeks Index to Stz after Stz 1 2 3 4 5 6 7 8 Glycemia 6.919.4 19.5 19.6 20.0 18.7 18.7 19.7 19.7 20.3 mM/l Body Mass, g 200 220210 200 210 210 210 220 220 230

TABLE 26 Change in Glycemia and body mass after administeringStreptozotocin to rat #16 not subjected to transplantation (control)Prior 4 weeks Weeks Index to Stz after Stz 1 2 3 4 5 6 7 8 Glycemia 6.725.1 25.5 27.6 27.6 25.6 22.9 25.0 22.2 24.1 mM/l Body Mass g 230 220210 200 190 200 190 180 180 180

TABLE 27 Change in Glycemia and body mass after administeringStreptozotocin to rat #25 not subjected to transplantation (control) 4weeks Weeks Index Prior to Stz after Stz 1 2 3 4 5 6 7 8 Glycemia 6.427.3 24.5 22.6 27.7 26.1 27.9 e.i. mM/l Body Mass, g 210 220 200 190 190180 180

TABLE 28 Change in Glycemia and body mass after administeringStreptozotocin to rat #26 not subjected to transplantation (control)Prior to 4 weeks Weeks Index Stz after Stz 1 2 3 4 5 6 7 8 Glycemia 5.817.7 17.3 18.3 22.9 21.3 19.9 18.3 19.7 17.7 mM/l Body 230 250 260 260250 270 280 280 280 300 Mass, g

Example 4 Cultures of Islet Cells

Cultures of islet cells were obtained in accordance of our designedtechnique from pancreases of 1-2 day newborn rabbits. For eachtransplantation, a culture containing approximately 200,000-300,000beta-cells was used. Immediately before transplantation, culture wascleansed from previously added growth medium embryonic serum andcollected it into sterile plastic test-tubes, each of 5 ml of volume.The attached fraction of a culture was removed with the help of specialcellular scraper (firm Corning-Costar). The floating fraction wasseparated by centrifuging of culture broth that covered the bottlebottom where the attached fraction was located. Final volume of cellularsuspension corresponded to the number of xenotransplantation operationsscheduled for the day, and each milliliter of suspension should containapproximately 100,000 islet cells (generally beta-cells).

Example 5 Insulinotherapy

For subcutaneous injection we used specific action insulin Aktrapid HMthat was administered twice a day: 9:00 and 19:00. Dose was selectedindividually. Criteria of successfulness of applied insulinotherapy wasdecreasing of hyperglycemia for up to 10 mmol/l and lower.

Example 6 Administering Islet Cells Cultures

Cellular suspension was injected by syringe through injection needle ofbig diameter, puncturing abdominal wall without anesthesia. To avoidinjuries to internal organs and vessels of a rat, a rat was turnedupside down, by that assuring anti-displacement of abdominal organs andformation of free space. After puncture of peritoneum the cellulartransplant was injected into this free space.

Cavity operation is needed for intra-splenetic administration, so fornarcosis by means of intraperitoneal injection we used preparedex-tempore Hexenal solution based on ratio of 50-60 mg per 1 kg of bodyweight. Transplantation into spleen pulp was performed as follows:median incision along abdominal white line opened abdominal cavity.Spleen was drawn out into the operation wound surrounding it by sterilewipes. Cellular suspension collected the day before was drawn intosyringe (2 ml volume) and through injection needle (0.5 mm diameter)administered into spleen pulp and into subcapsular zone. To avoidbleeding the injection mark was closed by medical glue MK-6.

Example 7 Laboratory Examination/Investigation

Capillary Blood Glucose was determined in experimental animals usingGlucometr Smart Scan 2-3 times a week. Serum insulin was detectedthrough immunoenzyme method, using sets for rabbit (human) insulin andcustom-made sets for specific detection of rat insulin.

Example 8 Histological Research

For studying dynamics of morphological alterations happening in isletsunder influence of various types of treatment of experimental diabetesmellitus, pancreases of animals killed at the specified times during theexperiment was fixated in freshly-made Buena mixture. After specifichistological procedures (washing off fixative, dehydration, etc.)fragments of pancreatic tissue were sealed into paraffin. Then, made bymicrotome microscopic sections (5-7 mkm thick) were dewaxed and coloredby hematoxilin and eosin as well as by aldehyde-fuchsine, for thepurpose of isolation of beta-cells.

For detection of fate of beta-cells transplanted into spleen pulp ofdiabetic rats, during specified times after transplantation, deadanimals' spleens and its fragments, which, judging from the scheme ofthe operation and some external signs, could, supposedly containtransplant, were exsected and immediately fixated in the Buena mixture.In succession as described above, paraffin sections were prepared andcolored in specified manner.

Example 9 Research Results: Characteristics of Culture

Islet Cells cultures obtained for subsequent transplantation conformedto the accepted functional criteria. As such, studying under invertedmicroscope demonstrated that by the 2-week cultivation term the cultureattained did not contain cells of exocrine tissue of pancreas thatballast cellular elements are absent includingleukocytes-“passengers”—initiators of rejection of islet cells after itsxenotransplantation. This data, and previous experience oftransplantation treatment of experimental diabetes mellitus, assured usthat there was no need for administering any type of immunosuppression.Control tests of insulin content in cultural broth demonstrated thatexisting beta-cells have high insulin-producing activity (Table 29).

TABLE 29 Indexes of basal and stimulated (25 mmol/l glucose) insulinsecretion (concentration mkUNIT/ml) in islet cells culture produced frompancreases of newborn rabbits: Sample # Basal Stimulated 1 2360 3330 211080 13450 3 7800 8760 4 5580 7650 5 7530 9900 6 8800 12300 7 5090 6850

Example 10 Results of Insulin Therapy

As it was stated in previous works, selection of effective doses ofinsulin in treatment rats with experimentally induced Streptozotocininduced diabetes mellitus is a difficult, lengthy (sometimes practicallyendless) and ungrateful work. Insulin-resistance often revealed in that.However, in majority of animals of the 1^(st) test group (5 out of 8) wesucceeded by means of high doses of insulin to lower hyperglycemia up tolevels of less than 10 mmol/l and to maintain such compensation ofimpairment of carbohydrate metabolism during the 8-week period (tab0.30).

TABLE 30 Changes in Glycemia in rats with experimentally induceddiabetes mellitus (1^(st) group) under the influence of daily insulintherapy. ## Length of insulin therapy (days) RAT 0 7 13 21 28 35 41 4653 60  9 17.7 15.5 12.1 10.8 11.9 10.0 7.9 8.5 6.7 6.4 10 28.7 29.0 26.722.4 27.8 23.3 22.8 24.9 15.5 18.4 11 18.6 18.9 15.9 11.0 9.7 12.3 11.58.6 10.6 9.7 12 16.1 12.0 11.1 8.7 6.7 5.4 6.6 8.1 6.3 6.3 13 29.7 26.722.8 13.4 14.5 16.9 18.5 14.6 20.3 18.8 14 24.4 22.2 18.0 22.1 18.3 19.117.7 14.8 18.8 14.3 15 19.0 18.7 14.3 11.2 8.6 9.9 10.8 8.6 9.9 7.7 1624.2 18.8 13.8 9.9 12.0 10.2 9.7 6.7 8.6 8.9 Average dose 0 4 8 12 1614.1 12.8 17.6 11.9 13.3 (unit) of insulin

Although we succeeded in lowering level of glycemia in rats #10, 13 and14, lack of adequate reaction in response to introduction of immensedoses of insulin (up to 20-30 units per day) did not allow us to revealtendency to normalization of glycemia and to admit presence in theseanimals of high individual insulin-resistance; and possibility forsignificant regeneration of own islet apparatus would be unlikely, allthe more so that after stopping administration of insulin all rats fromthe 1^(st) group had quick recurrence of hyperglycemia close to initiallevel.

Example 11 Results of Xenotransplantation of Cultures of Islet Cells

Expressed anti-diabetic effect of xenotransplantation of cultures ofislet cells was noted both in cases of injection of transplant intoperitoneum (2′¹ group of experimental rats), and in its introductioninto spleen pulp (3^(rd) group). Effect is seen in decreasing ofclinical manifestation of diabetic status and also in expresseddepletion of level of hyperglycemia. As data presented in tables shows,character of depletion of glycemia in the course of both methods ofadministering of xenotransplant is, to some extent, different from eachother. These differences comprise as in term of manifest ofsugar-lowering effect, its severity, and in its persistence (stability).

TABLE 31 Changes of glycemia in rats with experimental diabetes mellitusafter intraperitoneal xenotransplantation of cultures of islet cells(2^(nd) group) Before Rat transplan- Days after transplantation # tation2 7 10 13 15 17 20 22 1 24.1 18.7 12.2 15.5 12.0 11.9 14.5 12.4 14.8 216.6 15.0 13.0 8.8 6.8 9.0 7.7 6.7 9.1 3 29.0 27.9 16.1 17.7 22.2 18.415.5 14.1 16.5 4 21.2 18.8 12.2 13.0 13.0 11.0 12.2 10.5 8.9 5 17.9 14.815.5 16.8 15.9 16.0 15.9 14.0 13.7 6 21.9 19.1 14.2 12.8 11.1 8.5 9.07.8 7.1 7 17.1 16.6 11.4 9.8 7.0 7.9 6.4 8.1 8.6 8 25.6 21.1 17.7 17.118.8 20.7 18.6 21.2 20.0 M 21.7 19.0 14.1 13.9 13.3 12.9 12.3 11.9 12.3

TABLE 32 Changes of glycemia in rats with experimental diabetes mellitusafter intraperitoneal xenotransplantation of cultures of islet cells(2^(nd) group) - continued Days after transplantation Rat # 24 26 28 3032 35 37 40 42 1 13.2 13.9 12.5 14.4 16.8 12.2 15.5 12.0 11.9 2 7.8 7.07.4 6.7 5.5 6.4 6.4 5.9 5.5 3 17.2 14.8 12.5 14.7 15.6 14.1 16.5 16.117.7 4 10 10.1 9.2 10.5 8.4 7.9 11.1 8.7 9.0 5 13.9 14 15.9 12.4 14.715.0 14.3 13.8 15.2 6 8.1 8.0 7.0 6.7 7.1 8.4 9.0 7.8 8.1 7 8.7 7.4 6.45.1 6.5 5.6 4.9 5.8 6.1 8 18.8 20.6 21.8 22.2 19.2 18.6 21.2 20.0 17.8 M11.9 12.0 11.6 11.6 11.7 11.0 12.4 11.3 11.4

TABLE 33 Changes of glycemia in rats with experimental diabetes mellitusafter intraperitoneal xenotransplantation of cultures of islet cells(2^(nd) group) Days after transplantation Rat # 44 46 49 51 53 55 57 601 12.5 14.4 16.8 18.2 17.5 16.7 15.6 14.9 2 6.7 5.5 7.0 7.4 6.7 6.8 6.05.7 3 15.5 14.1 16.5 16.8 15.9 17.7 15.9 16.0 4 10.0 11.0 9.2 10.5 8.97.9 8.3 9.4 5 14.8 15.5 16.8 15.9 16.0 14.6 15.7 17.0 6 8.5 8.0 7.8 7.16.7 7.4 6.4 5.1 7 7.1 6.6 5.1 6.8 7.0 7.9 6.4 5.1 8 17.7 17.1 18.8 21.919.1 21.0 20.9 17.8 M 11.6 11.5 12.3 13.1 12.2 12.5 11.9 11.4

After analyzing the obtained results of measuring glycemia in rats withexperimental diabetes mellitus in which an intraperitonealxenotransplantation of cultures of islet cells was performed, it ispossible to made a conclusion of achieving expressed anti-diabeticeffect in majority of animals-recipients.

As such, rats #2, 4, 6, and 7 had remission of the diseases manifestedin stabilization of blood sugar at a normal or almost normal level.

Rats #1 and 3 demonstrated not so intensive decrease in glycemia level,although statistical data is very reliable. In such cases we may onlytalk about fractional remission of hypoglycemic status, all the more sothat rat #1 during the last weeks of observation revealed a tendency ofgrowing of previously moderate hyperglycemia.

In other animals (rats ##5 and 8) decline of glycemia was notsignificant and not stable, which fact bears evidence of a failure ofthese two transplantations of cultures of islet cells.

TABLE 34 Changes of glycemia in rats with experimental diabetes mellitusafter intra-splenetic xenotransplantation of cultures of islet cells(3^(rd) group) Before Rat transplan- Days after transplantation # tation2 7 10 13 15 17 20 22 17 17.1 15.5 16.8 13.5 12.2 9.9 12.4 10.2 9.4 1822.1 21.0 19.5 17.8 18.0 15.6 17.1 12.6 12.1 19 21.2 21.9 17.6 18.7 16.214.4 11.8 12.2 10.5 20 26.9 21.8 14.2 13.0 12.8 11.8 12.2 10.5 13.0 2121.9 14.8 15.5 14.1 15.9 13.6 15.5 14.0 13.3 22 28.8 29.1 24.2 30.8 25.921.8 25.2 27.1 22.7 23 15.5 13.6 11.5 8.8 9.0 9.7 7.4 8.0 6.6 24 22.623.1 17.1 14.7 15.8 12.2 15.5 12.0 14.2 M 19.3 20.1 17.1 16.2 15.2 13.614.0 13.3 12.5

TABLE 35 Changes of glycemia in rats with experimental diabetes mellitusafter intra-splenetic xenotransplantation of cultures of islet cells(3^(rd) group) - continued Days after transplantation Rat # 24 26 28 3032 35 37 40 42 17 10.2 10.9 10.0 9.4 8.7 11.2 10.5 9.8 9.9 18 11.8 8.77.4 6.4 5.1 6.5 6.6 7.9 5.7 19 17.7 16.8 11.2 14.0 10.6 11.4 10.6 11.19.1 20 8.4 9.0 8.8 8.7 7.5 8.4 9.0 9.7 9.0 21 15.9 12.4 13.5 12.4 14.015.1 13.4 13.8 12.8 22 18.1 18.0 17.0 18.1 18.4 17.9 18.1 17.8 18.1 239.7 7.4 7.5 6.5 5.4 5.6 5.4 5.8 6.6 24 18.8 20.6 21.8 22.2 19.2 18.621.2 20.0 17.8 M 13.8 12.1 12.2 12.2 11.1 11.8 11.9 12.0 11.1 Days aftertransplantation Rat # 44 46 49 51 53 55 57 60 17 11.5 10.4 8.1 8.2 8.76.7 6.6 4.9 18 6.7 5.5 7.0 7.4 9.2 10.5 8.9 7.9 19 10.5 9.4 10.5 8.8 9.97.4 8.9 9.1 20 9.1 6.7 7.4 7.1 11.0 7.8 9.8 7.5 21 14.8 15.7 17.0 15.913.6 14.0 15.5 16.8 22 17.5 17.8 16.7 16.8 16.0 15.7 14.9 15.8 23 8.76.0 5.9 8.3 7.6 6.7 4.6 5.7 24 21.5 21.1 19.1 21.0 18.8 21.0 18.9 19.7 M12.5 10.3 11.5 11.7 11.9 11.2 11.0 10.9

After xenotransplantation of islet cells culture into pulp of a spleen,expressed digression of hyperglycemia was noted in majority of animals(7 out of 8). At that, practical normalization of glycemia happened in 5rats with experimental diabetes mellitus (##17-20, 23). Also, in oneadditional recipient (rat #21) level of blood glucose depletedsignificantly, but its level remained at average hyperglycemic level,and in the second half of the term of observation gradual increase ofglycemia was noted, which may be even evaluated as a definite recurrenceof high hyperglycemia. In two of the group's animals (#22 and 24) therewas no ample anti-diabetic effect achieved, although rat #22 reachedstatistically reliable decrease of glycemia, but it cannot be classifiedas a remission of diabetes.

As it was previously noted, we expect divergence in glycemia changes incomparison between intraperitoneal and intra-splenetic methods ofxenotransplantation, but it appeared not be very fundamental. For thepurpose of simplicity of comparison, we combined glycemia indicators in2′^(d) and 3^(rd) group of animals in dynamics into one table (#34).

TABLE 36 Dynamics of changes in glycemia (average, M) in rats of the2^(nd) group (intraperitoneal xenotransplantation) and in 3^(rd) group(intra-splenetic transplantation) in rats - recipients: Pri- or totrans- plan- Days after transplantation Group # tation 2 7 10 13 15 1720 22 2^(nd) 21.7 19.0 14.1 13.9 13.3 12.9 12.3 11.9 12.3 3^(rd) 19.320.1 17.1 16.2 15.2 13.6 14.0 13.3 12.5 Days after transplantation Group# 24 26 28 30 32 35 37 40 42 2^(nd) 11.9 12.0 11.6 11.6 11.7 11.0 12.411.3 11.4 3rd 13.8 12.1 12.2 12.2 11.1 11.8 11.9 12.0 11.1 Days aftertransplantation Group # 44 46 49 51 53 55 57 60 2^(nd) 11.6 11.5 12.313.1 12.2 12.5 11.9 11.4 3^(rd) 12.5 10.3 11.5 11.7 11.9 11.2 11.0 10.9

Subsequent to the completion of physiological portion of research,animals enacted in this experiment, were put to death painlessly. Atthat, blood samples were taken from experimental animals, and processedserum was tested for insulin (also rabbit's, and rat's). Shown below areresults of immunoenzyme analysis (enzyme immunoassay) of samples ofrats-recipients' blood serum, which analyses were performed utilizingvarious special sets. (Table 35).

TABLE 37 Content of xenogenous (rabbit) and own insulin in blood serumof rats subjected to transplantation of cultures of islet cells ofpancreases of newly-born rabbits: Rat # Rabbit Insulin; Rat InsulinTotal Insulin  1 16 7 23  2 16 52 68  3 45 11 56  4 23 50 73  5* — — — 6 57 53 110  7 21 48 69  8 9 20 29 17 23 60 83  18* — — — 19 17 134 151 20* — — — 21 4 24 28 22 6 27 33 23 5 74 79 24 0 7 7 *no verifiableresult due to intensive hemolysis (mass erythrocyte lysis) of serum.In addition, we analyzed blood samples obtained after completion ofexperiments from rats with Streptozotocinal diabetes, who had beensubjected to insulin therapy In this, serum was examined for human andrat insulin content (tab. 36) with utilization corresponding sets forimmunoenzyme analysis.

TABLE 38 Content of human and rat insulin in blood serum of ratssubjected to insulin therapy (1^(st) group). Rat # Human Insulin Ratinsulin Total insulin  9 194 17 211  10* — — — 11 258 24 282 12 79 23102 13 144 0 144 14 331 4 335  15* — — — 16 261 0 261 *verifiable resultwas not obtained due to intensive hemolysis

It is not simple to interpret the obtained results. Complexity ofanalysis is conditioned, first of all, by the fact that demonstrated intables data reflects, in particular, an insulin-producing function ofxenotransplanted rabbit beta-cells and own (rat) beta-cells (Tab. 8), orof injected human insulin and own rat insulin (Tab. 9) only at themoment of completion of multi-day experiment. Certainly, it would begreat to have data about dynamics of content of varied types of insulinin varied terms during the experiments. But extreme difficulty ofgetting non-hemolyzed blood from small lab animals in quantitiesadequate for preparation of needed volume of serum without causingserious trauma to animals, does not allow implementing it without riskof loosing such valuable lab animals. So, we would be acting on thebasis of the data that we have available.

It appears that in majority of cases (11 out of 13) rabbit insulin wasdetected in blood of rats-recipients, which is an unchallengeableevidence of presence in animals' blood of rabbit 9 i.e. xenotransplantedbeta-cells secreting into “new owner's” blood an insulin relevant to adonor. Its concentration fluctuates significantly from 4 to 57 pmol/l.

Segment of rat-recipients in which their own species insulin wasdetected is even more (12 out of 13). Fluctuations of its content arevery large—from 7 to 134 pmol/l. Distinctive from interpretation ofpresence of xenogeneic insulin in animals subjected toxenotransplantation of islet cells, it is impossible to give asingle-value estimate to the detection of one or the otherconcentrations of insulin secreted by beta-cells of lab rats withexperimental diabetes. It is possible to evaluate more or lessobjectively to obtained data, when considering proportion of severalinterrelated quantitative data, namely: interconnection of glycemialevel and of total insulinemia, ratio of rat and rabbit insulin (inrats-recipients) or human insulin (in rats subjected to injection of itspreparation), and also to try to correlate dynamics of glycemia fromstart to finish of observation with different (in species (specific)origin) insulinemia.

An analysis was conducted in selective, but characterizing for commonconsistent patterns, order, various correlations of glycemia indicatorsand concentration of insulin in lab animals' blood serum were reviewed.From each group 3 rats were selected with more representative results oflab exams, and will tabulate all data in one table (Tab. #37).

TABLE 39 Indicators of glycemia and insulinemia in some rats of the2^(nd), 3^(rd) and 1^(st) group of experimental rats with experimentaldiabetes mellitus. Rabbit Initial Final (human) Rat Total glycemiaglycemia insulin insulin insulin Rat ## mmol/l mmol/l pmol/l pmol/lpmol/l 2 16.6 5.7 16 52 68 4 21.2 9.4 23 50 73 8 25.6 17.8 9 20 29 1717.1 4.9 23 60 83 19 21.2 9.1 17 134 151 24 22.6 19.7 0 7 7 9 17.7 6.4194 17 211 14 24.4 14.3 331 4 335 16 24.2 8.9 261 0 261

The analysis of results drawn in the 2^(nd) experimental group(xenotransplantation of cultures of islet cells into peritoneum) wasdone. In both successful cases (rats #2 and #4), remission of diabeticstatus was accompanied by significant decrease of glycemia—from 16.6 &21.2 mmol/l to 5.7 & 9.4 mmol/l respectively. At that, at the very endof 2-month observation normalization of levels of insulinemia(respectively up to 68 and 73 pmol/l) mainly on account of restorationof insulin-producing activities of own beta-cells of rats-recipients (52& 50 pmol/l respectively), although share of insulin secreted byxenotransplanted beta-cells of newly-born rabbits appears to berelatively sufficient (16 & 23 pmol/l respectively). The third animal ofthat group (rat #8) with initially higher level of hyperglycemia (25.6pmol/l), demonstrated functioning of transplanted beta-cells (rabbitinsulin concentration 9 pmol/l) along with own beta-cells (concentrationof rat insulin—20 pmol/l). Total (basically on account of own efforts ofislets of recipient's pancreas) insulin production secured statisticallymeaningful drop in hyperglycemia (up to 17.8 pmol/l), however, it wasnot enough for achieving remission of diabetic status.

In the 3^(rd) experimental group (intra-splenetic xenotransplantation ofislet cells) rats ##17 and 19 were chosen as examples, they had initialand final glycemia very similar to same in rats ##2 and 4 from the2^(nd) group. However, if data on glycemia and insulinemia in rats #2and 17 appeared to be very close (respectively: initial glycemia 16.6 &17.1 mmol/l, ending glycemia—5.7 & 4.9 mmol/l, rat insulin—52 & 60p/mol/l), then in rats #4 and #19 upon identity of initial glycemia(21.2 & 21.2 mmol/l) and similarity of ending glycemia (9.4 & 9.1mmol/l), the total insulinemia happened to be very distinctive (morethan 2 time—respectively 73 and 151 pmol/l). At such, this distinctionwas, in general, due to differences in concentration of rat insulin(respectively 50 & 134 pmol/l).

In rats of the first group, due to super-intensive insulinotherapy, itwas possible to achieve normal or close to normal content of glucose inblood. Apparently, due to restoration of applicable disturbedmetabolism, conditions for partial restoration of pool of own beta-cellsof experimental animals emerged. However, the degree of achievedregeneration appeared to be deficient for this amount of regeneratedbeta-cells to produce quantities of insulin sufficient to affectsignificantly the development of experimental diabetes mellitus.

In some rats that were subjected to xenotransplantation of islet cellscultures and that demonstrated remission of diabetic status, onlypartial regeneration of beta-cells was noted (picture 6). However,anti-diabetic effect was, probably, provided by insulin-producingactivity of beta-cells of newborn rabbits successfully transplanted intorats-recipients. This is indicated by findings of research of content ofxenogenous (rabbit's) and own (rat's) insulin in blood. (table 8).

Research of pancreases of rats with experimental Diabetes Mellitus, inwhich significant anti-diabetic effect after xenotransplantation ofislet cell cultures of newborn rabbits was noted, revealed regenerationof beta-cells. In such, the stated restorative process was noted, as arule, exactly in Langerhans islets, precisely in places of theirlocalization.

Special coloring of tissue of pancreas of rats-recipients subjected to asuccessful xenotransplantation of islet cells cultures demonstrated thatregeneration of structures of Langerhans islets happened, generally, onaccount of beta-cells. At the same time the appearance of single pointbeta-cells (colored by Aldehyde-fuchsin) is noted outside of isletlocalization, which can indirectly indicate a possibility of generationof beta-cells out of out-of-islet structures, probably, duct epithelium.

Accordingly, based on results of conducted scientific experiments, it ispossible to make several general conclusions:

Streptozotocin causes general destructing effect on beta-cells ofpancreatic islet cells, but at the same time, directly or indirectly,leads to the loss of other cells.

It appears that regenerative process in affected Langerhans isletshappens, mainly, due to restoration of beta-cells pool.

Intensive insulinotherapy is not capable only due to normalization ofglycemic status to provide decently expressed rehabilitation process inpancreatic islets of rats with Streptozotocinal Diabetes Mellitus.

Islet cells cultures produced from pancreases of newborn rabbitsutilizing an original method, consist, principally, of beta-cellspurified from ballast cellular elements and have a very highinsulin-producing activity.

Both intra-peritoneal and intra-splenetic xenotransplantation of isletcells cultures to rats with experimental Streptozotocinal DiabetesMellitus, in majority of cases, secures stable remission of diabeticstatus during at least 8 weeks.

Post-transplantation sugar-reducing effect is assured both, byfunctioning of transplanted beta-cells and by insulin-producing activityof to some degree restored pool of beta-cells in islets of pancreases ofrats-recipients. This is supported by findings on fluctuations ofconcentrations of exogenous (rabbits′) and own (rats′) insulin in bloodof experimental animals.

Histological examinations of pancreases of experimental rats confirmedinsignificant role of intensive insulin-therapy and expressedstimulation of regenerative processes in islets of rats withStreptozotocinal Diabetes Mellitus after xenotransplantation of isletcells cultures.

Possibly, regeneration of beta-cells happens not only in the borders oflocalization of Langerhans islets, but also in certain structures ofout-of islet pancreatic tissue.

It is difficult to give interpretation of these results. Only oneworking version can be advanced—presence of decreased sensitivity ofinsulin receptors in rat #19 towards own insulin. Insignificant decreaseof hyperglycemia in rat #24 can be explained by lack, apparently, ofunsettled (or rejected due to immunologic incompatibility, or disposedof its insulin-producing activity as a result of phenomenon ofapoptosis) xenotransplanted beta-cells (rabbit insulin—0) and weakhormonal activity of own beta-cells of the animal (rat insulin—7pmol/l).

Exactly the above stated reduced sensitivity of insulin receptors (butexactly towards introduced from outside (synthesized human insulin) canexplain very high levels insulinemia (respectively 194, 331 and 261pmol/l) in rats of the 1^(st) group (##9, 14 & 16) who had beensubjected to daily insulin therapy. At that, concentration of owninsulin in these animals was low (in rats 9 and 14) or even not detectedat all. (rat #16).

It is a possibility, that presence in the blood of large amounts ofexogenous insulin prevented more significant activity of own beta-cellsof islets of pancreas of rats of the 1^(st) group. High concentration ofexogenous insulin, due to feedback rule, facilitated peculiar atrophy ofislets apparatus “disuse atrophy”. Not excluding that on the backgroundof super-intensive insulin therapy, whose regime cannot compete withnormal secretion of insulin by healthy endocrine pancreas, ratsdemonstrated expressed hypoglycemic episodes, which were curtailed byexcessive food intake (feeding was unlimited), which in itself increasedrequirements in administered insulin. We shall consider, naturally,pro-diabetic impact of whole group of hormones which concentration issharply increased in the process of development of hypoglycemic status,and an impact of variety of stress-inducing situations, such as bloodtaking, operative interventions, and etc.

In contrast with intensive insulin therapy, moreexact—hyper-insulin-therapy, anti-diabetic effect of xenotrasplantationof cultures of islet cells rides by secretion into animals-recipients'blood of amounts of insulin being secreted by transplanted beta-cellsmore or less adequate to level of glycemia. In addition, secretion ofinsulin happens in abdominal cavity or in the spleen, resulting inhormone getting into system of portal vein, which can be considered aspractically physiological, i.e. natural way for organism.

Successful experimental researches that demonstrated high anti-diabeticeffect of grafting of islet cells obtained from pancreases of newbornrabbits, allowed to use such cultures in clinical practice.

Example 12 Intramuscular Xenotransplantations of Cultures of Islet Cells

Total of 112 patients with diabetes mellitus of type 1 were underwell-documented dynamic observation. Of 112 patients, there were 58 men,and 54 women. Patients' age at the moment of transplantation was from 16to 53—average 33.5 years old.

It is known that severity of manifestation of secondary diabeticcomplications depends significantly on longevity of the disease.Supposedly, destruction of own beta-cells of the patient as a result ofautoimmune process approximately happens on the 5^(th) year aftermanifestation of the disease. Secondary diabetic complications manifestitself usually in patients with longevity of disease of more than 10years. Because of that, all patients were divided in 3 groups inreference to longevity of the disease: a.) 1 to 5 years—16 people; b.) 6to 10 years—43 people, c.) more than 10 years—53 patients. All patientshad been examined to determine character of development of Diabetesmellitus and establishing presence of diabetic complications.

Usually, Islet Cells cultures received through the above-describedmethods out of 50-60 pancreases of 1-2 day newborn rabbits were used fortransplantation dose for 1 patient.

Each dose of the culture contained 1.5-2.0 min of beta-cells. Collectedimmediately prior to transplantation islet cells cultures were injectedthrough the syringe into muscles recti abdominis under local anesthesia.No immune suppression was used.

Below is the description of technique of transplantation of cultures ofislet cells.

One dose of cultures of islet cells represents a sterile suspension inHank's salt solution (10-15 ml volume) placed in a plastic tube markedin an appropriate way. Using an injection needle of not less than 7 cmin length and more than 1 mm in diameter, the islet cell suspension iscollected into a syringe of 20 ml in volume.

On the right side of the patients' umbilicus in the projection zone ofmusculus abdominis rectus utilizing separate syringe and correspondinginjection needle the local infiltration anesthesia of frontal abdominalwall (by Novocain or other anesthetic) is performed.

After anesthesia we use injection needle to perform puncture ofsub-aponeurosis space of the musculus abdominis rectus, and suspensionof cultures of islet cells The place of injection is to be closed by asterile bandage.

In addition to traditional administering of Islet cells cultures intotransverse abdominal muscle, a more complex but more physiologicalmethod of transplantation has found its application—transplantationthrough the portal vein, access to which is actualized throughbougienage of obliterated umbilical vein. There are some grounds toconsider (almost 20 of such transplantations were fully analyzed) thatdue to this method of administration, the quicker and more expressedsugar-reducing action of transplantation is achieved, as well assignificant reduction of requirements in exogenous insulin. (Shumakov etal., 1993 {16}.) However, the degree of therapeutic effect ofintra-portal transplantation of cultures of islet cells of pancreases ofnewborn rabbits on secondary diabetic complications is practicallyindistinctive from effects of intramuscular transplantation. Because ofspecific technical complexities and possible surgical risks of thismethod, it has been abandoned, and almost all transplantations areperformed utilizing a safe and simple method of injection cellsuspension under the aponeurosis of transverse abdominal muscle.

At the same time, while performing series of intraportaltransplantations, we examined an ability of beta-cells containing incultures of islet cells of newborn rabbits to respond to correspondingstimulus by secretion in-vivo—i.e. in an organism of a type-1 diabetespatient. Five male patients were subjected to this research, their agevaried from 25 to 45 years, history of disease—from 12 to 25 years.Along the guide, control of X-ray screen televisual apparatus, throughthe left subclavicular vein the catheter was placed in the right hepaticvein. Blood intake (5 ml) was performed instantaneously out of hepaticand portal veins (through trans-umbilical catheter that had been placedduring the prior transplantation of islet cells culture. After that, forthe purpose of local stimulation of intraportally transplanted isletcells cultures of pancreases of newborn rabbits, some 20 ml of20%-glucose solution was injected into portal vein, in analogue with arate of 1 gram of glucose per 1 kilogram of patient's body weight, whichhad been used in intravenous load test. Blood draft from portal andhepatic veins was done in 1 min, in 5 min, in 15 min, in 30 min, and in60 minutes after administration of glucose. As blood exam for insulincontent showed, prior to stimulation, there was already a differencebetween insulin concentrations in portal vein and in hepatic vein(respectively 4.9±0.6 and 6.1±1.0 mkUNIT/ml). In 1 minute after glucoseadministration insulin concentration was noted to increase 1.5 times inhepatic vein (from 6.1±1.0 to 9.1±1.3 mkUNIT/ml; p<0.05), which is twiceas large than its concentration in portal vein (4.2±0.5 mkUNIT/ml). Bythe 5^(th) minute after stimulation, concentration of insulin in hepaticvein was returning to the initial level, and from the 15^(th) minute itwas decreasing significantly, which, presumably, indicatespost-stimulation depletion of insulin-producing function of newbornrabbits islet cells cultures implanted to the portal system of liver.

These results demonstrate a substantial insulin-producing ability (inresponse to stimulation by glucose) of beta-cells of islet cellscultures xenotransplanted into recipient's liver and its possibleability to function on the principle of “feed-back reaction”.

Proof of functioning of transplanted beta-cells of pancreases of newbornrabbits was demonstrated by this original (unique) method because it isyet impossible to detect production of insulin by transplant based onsecretion of C-peptide, due to lack of existence in the world of setsfor immune-radiological or immune-ferment identification of C-peptide ofrabbits.

Example 13 General Results of Xenotransplantation of Cultures ofPancreatic Insular Cells to Rats with Experimental Diabetes Mellitus

Ability of cultures of pancreatic insular cells, received through theoriginal method from pancreases of newborn rabbits, to survive andfunction in in-vivo environment, has been demonstrated by us inexperiments of xenotransplantation of such cultures to animals withexperimental Diabetes Mellitus.

Vistar Line male rats of body mass of 180-220 gm, regularly fed, wereused as experimental animals. Experimental Diabetes Mellitus wasprovoked by sub-dermal application of Alloxan (dosage 200 ml for 1 kg ofbody weight) or by sub-dermal application of Streptozotocini (dosage 60ml/kg). During experiments and control probes we used only rats withAlloxan or Streptozotocin-induced Diabetes, those whose level ofhypoglycemia on empty stomach was 20 mmol/l and higher. Earlierconducted tests indicated that such animals did not have spontaneousreversion of experimental Diabetes Mellitus.

After transplantation of pancreatic islet cells cultures, 88 out of 104rats with stable or severe Alloxan-induced Diabetes Mellitus (almost85%) displayed firm remission of diabetic status up-to the end ofexperimental term (20 weeks). Firm decrease of blood sugar levels ofalmost-up-to-normal levels was registered in blood ofanimals-recipients. At the same time, characteristic clinical symptomsof diabetes were also vanishing (such as weight loss, polydipsia,polyuria). Anti-Diabetic effect of Xenotransplantation was clearlydemonstrated both in cases of application of cultures into liver(through portal vein or directly into liver's parenchyma) and also intospleen (cultures were brought in intra-pulp.), and also through theabdominal muscles. Even after 8 weeks after Xeno-transplantation,pancreatic islet cells with preserved structure and with signs ofsecretory activity was detected in places of implantation in rats withremission of experimental Diabetes.

During special series of experiments the role of preliminary cultivationof pancreatic islet cells in vitro was demonstrated clearly in survivalof cells in organisms of xenogeny recipient. For that purpose, weperformed comparative analyses of results of xenotransplantation ofcultures of pancreatic islet cells of pancreas of human fetuses andxeno-transplantation of non-cultivated fetal Island tissue to rats withexperimental Diabetes Mellitus. It was detected that sugar-reducingeffect is more expressed and long-lasting in cases of transplantation ofpre-cultivated pancreatic islet cells in comparison with transplantationof non-cultivated tissue of Pancreas, which results only in short-livedremission of Diabetic status. So, the immune-modulating result ofcultivating in vitro was experimentally proven to significantly increasethe term of survivability in an organism of alien recipient.

Pancreases of 18 rats-recipients, on whom successful xenotransplantationof cultures of Pancreases of newborn rabbits had been performed, weresubjected to histological exam in 8 weeks after transplantation. Forthat purpose a fragment of pancreas was fixated in Buena solution andwas drown in paraffin. Slices (5-7 mkm thick) were colored byhematoxilin and eosin, and also by Aldehyde-fuchsine for revealing ofn-cells. At the same time, pancreases of 6 control animals who haduntreated alloxan-induced Diabetes as well as pancreases of 6 healthyrats (no Alloxan applied) were closely examined.

While examining pancreases of healthy intact rats, some 45 to 76% ofbeta—cells, as expected, were found in “Langerhans” islands. Rats withuntreated Alloxan-induced Diabetes had sharply decreased amount ofβ-cells in Islands—in average 8.3+−1.1%.

Significantly higher amount of beta-cells in islands was discovered inrats-recipients. In animals, who had been subjected toxenotransplantation of pancreatic islet cells cultures, their ownPancreases displayed typical β-cells and its share among “island’ cellswas from 10 to 55% (some from 7 to 21%) (average 23.5±8.8%).With regards to these experiments, we may assume that anti-diabeticeffect of xeno-transplantation of OK cultures on developments ofexperimental diabetes in rats is occurring 2 general ways: a.)Functioning of transplanted β-cells, confirmed, in addition to expressedsugar-reducing effect, also by revealing groups of transplantedpancreatic islet cells in the pulp of spleen of animals-recipients; b.)Stimulating effect of transplantation of Pancreatic islet cells cultureson the Island apparatuses of pancreas of rats-recipients, whichpossibility is confirmed by data of histological exams revealingexistence of significantly frequent of Islands with normal β-cells andbigger share of them in Islands of pancreas of rats-recipients than ofrats with untreated Alloxan-induced Diabetes. Successful experimentalresearch became grounds for performing clinical transplantation of isletcells of pancreas of newborn rabbits to Diabetes-type-1 patients.

Example 14 Clinical Transplantation of Cultures of Pancreatic IsletCells Produced from of Pancreases of Newborn Rabbits

Total of 112 patients with Type-1 Diabetes Mellitus (Insulin-dependentdiabetes mellitus—IDDM) were under well-documented dynamic supervision.Of total of 112 patients there were 58 men, and 54 women. Patients' ageat the moment of transplantation varied from 16 to 53—average 35 yearsold.

It is known that severity of manifestation of secondary diabeticcomplications depends significantly on duration of the insulin-dependentdiabetes mellitus. Supposedly, destruction of own β-cells of the patientas a result of autoimmune process approximately happens on the 5^(th)year after manifestation of the disease. Secondary diabeticcomplications manifest itself usually in patients with duration ofdisease of more than 10 years. Because of that, all insulin-dependentdiabetes mellitus patients were divided in 3 groups in reference toduration of the disease: a.) 1 to 5 years—16 people; b.) 6 to 10years—43 people, c.) more than 10 years—53 patients. All patients hadbeen examined with the aim to determine character of development ofinsulin-dependent diabetes mellitus and establishing presence ofdiabetic complications.

Usually OK cultures received through the above-described methods out of50-60 pancreases of 1-2 day newborn rabbits were used fortransplantation for one patient. Suspension was usually, delivered intotransverse abdominal muscle under local anesthesia. No immunesuppression was used.

Below are the results of xenotransplantation of pancreatic islet cellscultures on the course of development of insulin-dependent diabetesmellitus, on expression of its complications in patients of differentduration of the disease.

Transplantation of Cultures of Pancreatic Islet Cells of Newborn Rabbitsto Patients with Insulin-Dependent Diabetes Mellitus from 1 to 5 Years

5 out of 16 patients of this group had insulin-dependent diabetesmellitus with sharply labile character. 2 of them had frequent (severala week) spontaneous (without known provoking reasons) hypoglycemicconditions, which caused numerous inpatient treatment attempts, but allattempts to stabilize course of disease or to determine insulin dosagewere to no avail. 3 patients had labile insulin-dependent diabetesmellitus with possibility of development hardly-eliminating ketoses;attempts to reach metabolic compensation yield only short-lived effect.

Three patients (2 of them with labile Diabetes Mellitus) revealedsymptoms of sensori-motor neuropathy—paresthesia and pulling pain incalf—muscles. After intramuscular xenotransplantation of pancreaticislet cells cultures, majority of patients noticed reduction in usuallyelevated day-average glycemia levels during 2-4 weeks. Retention of itsvalue within the range corresponding to good compensation ofcarbohydrate metabolism (average 7.8 to 9.9 mmole/kg) was noted furtherduring at least 12 months of post-transplantation supervision. On this,in all 5 patients with labile IDDM the course of the disease acquiredstable nature: predisposition to hypoglycemic conditions and ketosisdisappeared. Improvement of glycogenic control after transplantation ofcultures of pancreatic islet cells of newborn rabbits confirms theinformation on determination of glycozylated hemoglobin in recipients'blood (reduction from pre-transplantation 12.4% to 9.6%, 8.3 and 10.1%relatively to 6, 9—and 12 months after transplantation. Elevation ofinsulin-dependent diabetes mellitus compensation and clear tendency toreduction of average daily level of glycemia allowed us by the end ofthe 1^(st) month to somewhat reduce dosage of administered insulin(average 12%), which remained to some degree reduced in 3, 6, 9 and 12months after transplantation—relatively for 31.5%, 36.2%, 25.5%, and18.4%. On this, 3 patients' requirements in exogenous insulin decreasedbetween 4^(th) and the months after transplantation for more than 50%(from 54% to 86%), at the same time for 2 patients the doze ofadministered insulin by the 1-3 month of post-transplantation period wastemporarily (for 2-4 week term) somewhat increased for 13% and 12%.

Because in patients with history of the insulin-dependent diabetesmellitus of less than 5 years possibility of presence of own β-cellsexists, we would expect evaluation of residual secretion of C-peptideprior to transplantation (analyses were performed automatically with thehelp of immune-ferment method, which usual parameters of content ofC-peptide in blood serum is 0.5-3.5 ng/ml). It turned up that prior totransplantation only 3 out of 6 patients (19%) had no secretion ofC-peptide neither basal (on empty stomach) no stimulated (by standardbreakfast). Duration of their insulin-dependent diabetes mellitushistory was longer than 3 years. Average level of basal and stimulatedC-peptide in patients with disease duration from 1 to 5 years (includingzero exponents in 4 of them) accounted for relatively 0.12 and 0.36ng/ml. After transplantation in 2 out of 3 “C-peptide-negativerecipients” concentration was registered—first stimulated, then (by the3^(rd) month)—a basal secretion of C-peptide, which indicated arestoration of insulin secretion by own β-cells of the patient.“C-peptide-positive recipients” demonstrated substantial increase ofC-peptide content in blood serum, which in 5 patients even reached thenormal factor. It is important to point that by the end of the 1^(st)year of observation there was no expressed tendency to depletion ofC-peptide in recipients' blood. Such were the changes noted during the12 months after the first transplantation to insulin-dependent diabetesmellitus patients with history of disease duration of 1 to 5 years.

Nine patients from this group with average interval of 13.3±1.8 monthswere subjected to repeated intramuscular transplantations of pancreaticislet cells cultures of newborn rabbits: 1 patent three times, 3patients—twice, and 5 patients—one time. No one of these patientsdisplayed neither local nor general signs of rejection/disengagement ofa transplant, nor any allergic reactions.

Eight out of 9 patients, who had been subjected to repeatedtransplantation revealed therapeutic effect of no lesser value than ininitial transplantation. In 3 of out of 4 patients, who were subjectedto xenotransplantation of pancreatic islet cells cultures thrice (i.e.+2 repeated times) increment of clinical effect was noted, such asincrease in muscle mass and significant improvement of life tonus. Inthis, stable course of insulin-dependent diabetes mellitus preserved,and symptoms of secondary diabetic complications were absent. The onlyone patient in this group who sustained 4 transplantations (by the endof this experiment history of his disease was longer than 9 years)during the 5,5, years of observation demonstrated no signs ofdestabilization of the course of disease (prior to 1^(st)transplantation it was sharply labile), and still no signs of diabeticangiopathy. It seems like accrued effect of repeated transplantations isgoverned by increase in secretory activity of own Island Apparatus ofrecipients confirmed by increase of concentration of both basal andstimulated human C-peptide with each transplantation.

Transplantation of Cultures of Pancreatic Insular Cells of NewbornRabbits to Patients with Disease History of 6 to 10 Years.

Total of 43 insulin-dependent diabetes mellitus patients with durationof disease from 6 to years were under supervision (average duration 7.8years); 24 men and 19 women. Age of patients at the moment of firsttransplantation was in the range from 15 to 43 years (average 28.3). 12patients had a sharply expressed labile character of the disease, whichhad not been stabilized during several attempts of inpatient treatment.16 patients had secondary symptoms of insulin-dependent diabetesmellitus complications; 9 of them had only sensori-motor neuropathy anddeveloping nephropathy (Mogensen stage 3), 2 patients—had autonomousneuropathy (predisposition to tachycardia), and 2 recipients—developednon-proliferating retinopathy.

In 1-2 months after xenotransplantation all 12 patients with priorlabile insulin-dependent diabetes mellitus status changed to morecontrolled and manageable status. Usually, stabilization of indexes ofcarbohydrate metabolism occurred also. As such, prior totransplantation, recipients' daily average blood glucose was fluctuatingbetween 9.6 to 14.1 mmol/l, but in 1 month after transplantation thedaily glycemia was 6.6. to 11.2 (average 8.6 mmol/l). Maximal reductionof this index was noted by the 3-month term (7.8 mmol/l), but by the endof the 1 year term it still remained satisfactory—at a level of 8.8mmol/l).

Improvement in compensation of carbohydrate metabolism was confirmed bychange of amount of glycosylated hemoglobin in patients' blood from 12%prior to transplantation (average) its level in 3 months was already10.8%, in 6 months—9.4%, in 9 months—somewhat increased (to 10.9%), butby the year's end reduced again to 9.8%.

Also, in majority of patients in this group a forced decrease of dailyadministered insulin doze in comparison with the pre-transplantationlevel: in 3 months after the transplantation it decreased in average for15.22%, in 6 months—for 30.1%, in 9 months—27.0%, in 12 months—25.1%.

Despite significant insulin-dependent diabetes mellitus history in thispatients' group (from 6 to 10 years—in average 7.8), only approximately75% (32 patients) prior to transplantation had had no own insulinsecretion (C-peptide completely absent). Because other patients in thisgroup had basal level of C-peptide varied from 0.05 to 0.2 ng/ml, andstimulated—from 0.1 to 0.3 ng/ml, its average concentration in the groupbefore transplantation was on empty stomach—0.07 and afterstimulation—0.08 ng/ml. After transplantation, steady increase ofC-peptide concentration in blood was occurring—in 3 months basal levelwas in average same 0.07 and stimulated level—already 0.11 ng/ml; in 6months—more than 3-time increase—relatively up to 0.38 and 0.43 ng/ml,but by the 9^(th) month—decrease up to 0.09 and 0.13 ng/ml.

During post-transplantation period, there were signs of more positivecourse of secondary complications in this group's recipients. Symptomsof both—sensori-motor and autonomous neuropathy started to weakenalready by the end of 1-1.5 month after xeno-transplantation, and almoststopped bothering patients by the 3′ month. Also, in both patients withdiabetic nephropathy a protein-urea disappeared completely by 2-4^(th)month after transplantation, and it did not reoccur for almost a yearafter the transplantation. Patients with diabetic retinopathy did notregister increase in pathological changes in fundus of the eye.

Repeated transplantations (in 7-13 months after the 1^(st)transplantation) were performed on 14 patients: 1 time on 5 patients,twice on 5 patients, three times on 4 patients, and 4 repeatedtransplantations—on 1 patient. In majority of cases repeatedtransplantations of pancreas insular cells, in minimum, contributed topreservation of positive changes in patients' status that had occurredafter the 1^(st) transplantation. Specifically noted shall be the resultof successive (interval 7-9 months) 5 transplantations in patient withsevere sensori-motor neuropathy, which had led 48-year-old man (withInsulin-dependent diabetes mellitus history of 8 years) to severedeconditioning (incapacitation/inability to wok) due to severe pain inextremities and expressed muscular atrophy, especially in lowerextremities. After 1^(st) transplantation pain in extremities lessened,and then after the 2^(nd) transplantation it completely vanished, andthen muscular tonus and volume began to regenerate. As a result ofperformed transplantation, during the 4.5 years of observation thepatient's muscular volume expanded for 23 kg, his muscular tonusnormalized, as well as conductance of nerve impulse through motornerves. Also, Diabetes acquired stable course; doze of introducedinsulin decreased for 50%. It is possible, that reduction of exogenousinsulin was significantly stipulated by significant revival of ownB-cells of the patient, as human C-Peptide content in recipient'sblood—0.1 ng/ml (empty stomach) and 0.1 ng/ml (stimulated) prior to thatbecame relatively 0.36 and 0.55 ng/ml by the end of the 4-year period ofpost-transplantation observation.

Transplantation of Pancreatic Insular Cells Cultures of Newborn Rabbitsto Patients with Disease History of More than 10 Years.

This group consisted of 53 persons—32 women and 21 men. Patients' age atthe moment of the first transplantation varied from 21 to 53—average33.4. Duration of disease varied from 11 to 27 years (average 14.8).

9 patients had true labile course diabetes: spontaneous hypoglycemicconditions often alternated with ketone-acidosis episodes.

There were secondary diabetic complications discovered in 38 patients:11 had only sensori-motor neuropathy; 1 patient had sensori-motorneuropathy and diabetic cataract, 5 patients—sensori-motor neuropathy,initial nephropathy (in Mogensen) and non-proliferating diabeticretinopathy; 8 patients—arising nephropathy and non-proliferatingretinopathy; 6 patients—expressed nephropathy and pre-proliferatingretinopathy; 2 patients—arising nephropathy and proliferatingretinopathy; 2 patients—expressed nephropathy and proliferatingretinopathy; 2 patients—sensori-motor and autonomous neuropathy,expressed nephropathy and pre-proliferating retinopathy, and 1patient—sensori-motor and autonomous neuropathy, uremic stage ofdiabetic nephropathy and proliferating retinopathy.

As such, diabetic neuropathy was revealed in 23 patients, including 20with sensori-motor and 3—with autonomous. Diabetic nephropathy wasrevealed in 24 patients, including arising nephropathy in 15 patients,expressed—in 8 patients, and uremic stage in 1 recipient. Diabeticretinopathy was revealed in 26 patients, including non-proliferatingstage—in 13 patients; pre-proliferating stage—in 8 patients, andproliferating stage—in 5 recipients.

With the purpose of diminishing of danger of development of hypoglycemicconditions, patients with expressed stages of diabetic retinopathy andnephropathy were transplanted a dose of pancreatic islet cells culturesreceived from no more than 40 pancreases of newborn rabbits. Allrecipients with initially labile IDDM course had relatively quick(during 1-3 months) stabilization of level of glycemia, and moreadequate regime of insulin-therapy was chosen.

There was a substantial reduction of average level of daily glycemia inpatients of this group: from 12.8 mmol/l prior to transplantation to 9.8mmol/l in 3 months after transplantation, and 10.1 mmol/l in 9 monthsafter transplantation. In correspondence to changes in glycemiaglucozylated hemoglobin concentration also reduced in recipients' bloodfrom 13.1 to 10.0%.

Significant increase of degree of compensation of IDDM was accompaniedby decrease in requirements of recipients in exogenous insulin. By the3^(rd) months after xeno-transplantation, doze of administered insulinwas reduced for patients of this group in average for 12.5%, in 6months—for 26.6%, in 9 months—for 25.0%, in 12 months—only for 9.8%,which demonstrated that requirement for exogenous insulin topre-transplantation level. Maximum reduction was noted in Patient C.(female) (26 years old, duration of IDDM—20 years; secondarycomplications—sensori-motor neuropathy, expressed nephropathy,pre-proliferating retinopathy). Already in 2 weeks after intra-musculartransplantation of pancreatic islet cells cultures received out of 40pancreases of newborn rabbits, reduction in need for administeredinsulin was as such—it dropped from 36 to 24 units/day, in 6 weeks—to 16units/day, in 10 weeks to 4 units/day (i.e. 90% reduction). At the sametime on the background of stable condition, the level of daily averageglycemia did not exceed 9 mmol/l. HbA1c content in 4 months aftertransplantation reduced to 8.7% and did not exceed 9% during the periodof at least 1.5 years. At the same time, substantial reduction ofexpressiveness of secondary diabetic complications also occurred.

4 patients, despite their long history of IDDM (from 10.2 to 13.5 years,average 11.1 years), revealed residual secretion of C-peptide (inaverage 0.05 ng/ml on empty stomach, non-stimulated. However, aftertransplantation by the 3^(rd) month the amount of C-peptide-positivepatients doubled up—and these 8 patients (average duration ofdiabetes—12.6 years) concentration of C-peptide on empty stomach was inaverage of 0.09 ng/ml, and stimulated—0.12 .ng/ml.

Effect/influence of transplantation on degree of manifestation ofdiabetic complications depended in majority of cases on its types andclinical stages (advancement). So, if all patients with sensor-motorneuropathy had substantial improvement of the course of thiscomplication already after the first transplantation of pancreatic isletcells cultures, but 2 out of 3 patients with autonomous neuropathy onlysecond transplantation made any positive effect.

12 out of 15 patients with initial stage of diabetic nephropathy hadfirm disappearance of micro-protein-urea and elimination of tendency toarterial hypertension. Positive post-transplantation effect was observedin patients with expressed nephropathy almost in 63% of cases—in 5 of 8recipients. At the same time, extraction of protein with urinesignificantly reduced: macro-protein-urea interchanged tomicro-protein-urea (less than 0.3 g/day) and a tendency to reduction andnormalization of elevated blood pressure, which allowed to significantlyreduce doses of hypotensive remedies or even to terminate administeringit. 2 out of 3 other recipients during the period of observation(relatively 2 and 2.5 years) there were no signs of progressive diabeticnephropathy.

A the same time a patient with the final stage of diabetic nephropathyhad only a short-lived positive effect: for 4-5 weeks afterxenotransplantation of cultures of pancreatic islet cells his moderatelyelevated level of blood creatinine and urea approached the upper borderof normal indexes, after which relatively slow but firm progression ofchronic kidney insufficiency took place.

10 recipients with initial and expressed stages of diabetic nephropathywere subjected to repeated transplantations of pancreatic islet cellscultures, and 4 of patients with expressed nephropathy became 3-timerecipients during the 3-4 year period. 9 out of 10 patients subjected tore-transplantation were noted for at least no further progression ofkidney function problems.

A patient with diabetic cataract (age 22 years old, IDDM) duration—11years) by the 5^(th) month after transplantation was examined byoculists who stopped noticing spots of clouding of the crystalline lens,they evaluated this change in clinical picture as <<cataractresorption”.

In 10 out of 13 patients (i.e. with non-proliferating retinopathy therewere no progression of pathological changes noted during the whole termof observation (from 1 to 6 years), with improvement of the eye funduspicture; in 5 recipients (absence of retina detachment, decrease ofamount of micro-aneurisms.) However, in 3 patients withnon-proliferating retinopathy there was increase in amount of aneurismsand regional hemorrhages occurred.

Out of 8 observed patients with pre-proliferating retinopathy, in 3cases after 2-4 years after the only xenotransplantation of pancreaticislet cells cultures, proliferating process was noted. At the same time3 patients of the same group (63%), who prior to transplantation had hadlaser-coagulation procedure performed, did not need repeated lasertreatment during the whole term of post-transplantation observation—from1 to 6 years. In this, 3 patients of this group had relatively 1, 2 and3 repeated transplantations with interval from 9 months to 1.5 years.

After xeno-transplantation of P.I.C. Cultures, 2 out of 5 patients withproliferating stage of diabetic retinopathy had relatively long(duration 1 and 1.5 years) stabilization of clinical picture of theeyeground with moderate increase of visual functions (probably, due toactive resorption of hemorrhages), and further progression ofproliferating process was noted, herewith 1 patient (female) hadrepeated hemorrhage into vitreous body with substantial worsening ofvisual functions.

Using transplantation of cultured islet cells produced from pancreasesof newborn rabbits appeared to be very effective in children's diabeticpractice. Late results of xenotransplantation of cultures of islet cellsin children with type-1 diabetes mellitus were researched throughobservation of 20 patients prior to transplantation and in 5 years afterthe first transplantation (Volkov, 2005 {1}). Comparative group (controlwithout transplantation) consisted of 20 children selected on principle“occurrence-control” with an allowance for age, sex, longevity of thedisease, level of compensation, requirement in insulin and developmentof complications.

Catamnesis analysis demonstrated that xenotransplantation of cultures ofislet cells has a positive affect on insulin requirements. Thus, alreadyby the 3^(rd) month after transplantation dose of administered insulindecreased in half of recipients, in comparison with the initial level,and by the end of the 1^(st) year—43% of patients had lesser requirementin insulin. There were no such changes in the comparison group.Dose-response effect was noted: the most decrease in insulinrequirements was noted after administration of the culture containingapproximately 5 min of beta cells. At that, in post-transplantationperiod more persistent and expressed compensation of carbohydratemetabolism, which is confirmed by dynamics of daily average glycemia incomparison to the control group. Already in 3 months afterxenotransplantation, level of daily glycemia reduced from 10.78±0.55mmol/l to 8.6±0.4 mmol/l against 9.15±0.72 mmol/l (initially 10.65±0.79mmol/l). In 1-year period after transplantation these distinctionsbecame more meaningful: in observation group daily average glycemia is8.5±0.39 mmol/l against 10.14±0.6 mmol/l in control group. Similartendencies remained in cases of repeated transplantations.

Therapeutic effect of transplantation of cultures of islet cells on thecourse of diabetic complications proved to be extremely important.Long-term clinical observations demonstrated that, in comparison withthe control group, there were less frequent occurrences of diabeticnephropathy, retinopathy, and growth disorders (as manifestation ofMoriak syndrome) in children with diabetes mellitus type-1 aftertransplantation treatment. Thus, decrease in occurrence of retinopathyfrom 25% to 11% (increase in this indicator from 23% to 25% in controlgroup). Loss of albumin with urine significantly decreased—from 207.4 to78.7 mg/day in patients with diabetic nephropathy afterxenotransplantation, whereas in comparison group this indicator keptgrowing—from 220.67 to 273.1 mg/day.

Such complication as Moriak Syndrome is one of the features ofchildren's and adolescent diabetology, growth disorders included in itsstructure. Usage of anabolic preparations for treatment of statedpathology leads to a short-lived effect, to advancement of closing ofgrowth zones and in reduction of final body height (growth). In thecourse of xenogeneic transplantation of cultures of islet cells,rapidity of growth was revealed but without change of speed in closingof growth zones. As such, share of patients having height lower than 5%(authentic nanism), decreased from 18% to 14% during the 1^(st) year ofobservation. In 5-year catamnesis, not one patient with height lowerthan 5 percentili among children of observation group was found. Incomparison group share of undersized children increased from 20% to 22%during the 1^(st) year of observation. In 5-year observation, share ofpatients with height under 5 percentili reduced to 10% due toapplication of intensified insulinotherapy.

Immunological research demonstrated that xenogenous transplantation ofcultures of islet cells does not cause substantial and long-termactivation of autoimmune process, which is very important in applicationof this type of therapy in patients with partially preservedinsulin-producing function. Careful observation of patients fromobservation group (with annual inpatient examination) did not revealcontamination of zoonosal infections, not even one.

Effect of Xenotransplantation of Cultures of Islet Cells of Pancreasesof Newborn Rabbits on Immunity Indicators in Patients with Type-1Diabetes Mellitus.

Complex immunologic research in 20 patients with type-1 diabetes wasperformed after completion of primary intramuscular transplantation ofcultures of islet cells of pancreases of newly-born rabbits, and in 12patients with type-1 diabetes after repeated xenotransplantations ofcultures of islet cells. 17 standard tests of cellular and humoralimmunity were used in immunologic research, systems of phagocytes andcomplement, including titer of complement-fixing antibodies towardsislet cells of pancreases of newly-born rabbits.

It is proven that in patients with type-1 diabetes prior totransplantation of Islet Cells cultures activation of T-cell immunitynexus was observed, as well as presence of imbalance of immunoregulatorysubpopulation of T lymphocytes, and activation of nonspecific protectionfactors. After transplantation 8 recipients (i.e. 40%) after the primarytransplantation of OK cultures showed low KFA titer of antibodiestowards total antigen OK and insulin, and 10 patients (50%) showedmoderate increase of B-lymphocytes count at 2-3^(rd) month, with itssuccessive reduction.

In determining subpopulations of T lymphocytes in dynamics afterxenotransplantation, there was a tendency to normalization of content ofT lymphocytes by the 7-10^(th) day. Amount of CD4-cells continued todecline, amount of CD3-cells continues to increase. By 14-20^(th) daythe number of helper T-cells measured up to normal. By the 2-3^(rd)month after transplantation, the on-going normalization of indexes ofcellular immunity was conjugated with achieving of good compensation ofthe disease.

Examinations of immune status in patients subjected to repeatedtransplantations of cultures of islet cells of newborn rabbits did notreveal any signs of immune response to xenotransplant. Quantitative andfunctional indexes of immunity were comparable to such in primaryxenotransplantations.

Xenotransplantation of Cultures of Islet Cells of Pancreases of NewbornRabbits to Patients with Recently Diagnosed Insulin-Dependant DiabetesMellitus.

Type-1 diabetes mellitus patients with history of disease of longer than5 years usually become recipients of OK cultures; they practically lackinsulin-producing activity of own islet apparatus of pancreas, and inmajority of cases secondary diabetic complications are advanced indifferent stages.

However, hopeful results were achieved also after intramusculartransplantation of cultures of islet cells of pancreases of newbornrabbits to patients with recently diagnosed diabetes mellitus type-1.

10 young people (average age 18±02 years) with average history ofdiagnosed diabetes (6±0.2 month). Positive results in glycemia controlwere noted in all patients during the 6-8 months after xenogenoustransplantation (fasting glycemia 5.2±0.2 mmol/l, after foodintake—7.0±0.15 mmol/l, daily glucosuria—0.25±0.03 grams, HbA1—5.8±0.2mmol/l). Daily dose of exogenous insulin was reduced from 0.62±0.11 to0.28±00.2 units at 1 kilogram of body weight. 7 patients (i.e. 70%cases) during the post-transplantation period had partial and 2recipients (i.e. 20% cases)—full remission of diabetic status (inducesby transplantation “honeymoon” of diabetes mellitus type-1), whichcontinued for 4-8 months. At that, increased in pre-transplantationperiod levels of glucagons and growth hormone in recipients bloodnormalized (glucagons concentration decreased from 112.0±2.1 to 69.7±1.2ng/ml, p<0.05; growth hormone—from 8.9±0.04 to 5.6±0.02 ng/ml, p<0.05)with successive normalization of daily profile of growth hormone. HumanC-peptide concentration in patients' serum increased from 0.3±0.01 to1.26±0.02 ng/ml; p<5; which attests to significant decrease of secretionof own insulin in recipients under the influence of performedtransplantation of xenogenous islet cells. At that, patients displayednormalization of indexes of cellular and humoral immunity, which hadbeen pathologically altered prior to transplantation (table 40)

TABLE 40 Indexes of immune status in patients with recently diagnosedIDDM after xenotransplantation of cultures of islet cells IgA IgM IgGMnths Tx OKT3 % OKT4 % OKT8 % OKB7 % OK1a % mg % Mg % mg % Rec 0 80 ± 5*50 ± 3* 30 ± 3  18 ± 2* 14 ± 2*  250 ± 13**  221 ± 18** 15008 ± 22*  n =10 1 80 ± 3* 51 ± 2* 29 ± 4 16 ± 3 12 ± 2  212 ± 9*  212 ± 10* 1472 ±14** 3 66 ± 2  36 ± 4  30 ± 4 11 ± 3 8 ± 3 174 ± 12 169 ± 10 937 ± 13  876 ± 4* 48 ± 2* 26 ± 3 14 ± 3 12 ± 2* 217 ± 13 224 ± 19 1461 ± 12**Control Healthy 68 ± 4  38 ± 5  30 ± 4 10 ± 2 7 ± 2 162 ± 16 151 ± 8 910 ± 12  OKT3—T cells; OKT4—T helpers; OKT8—T suppressors; OKB7—Blymphocytes, OK1a—anti-HLA-DR; Ig—immuno-globulins; *p < 0.05; **p <0.001 - in comparison with control

Following general changes are observed after xenotransplantation ofcultures of islet cells to patients of diabetes mellitus type-1:

-   -   Stabilization of course of labile forms of disease, which        results in successful selection of adequate insulinotherapy, and        to significantly increase the degree of compensation of impaired        carbohydrate metabolism;    -   Decrease in requirements for exogenous insulin for 20-30% in ⅔        of recipients;    -   Progressing of late diabetic complications suspends neuropathy,        nephropathy, retinopathy); involution of initial stages in more        than 80% cases.

It is rational to give explanations regarding possible mechanisms ofanti-diabetic effect of transplantation of cultures of islet cells ofnewborn rabbits. In spite of wide adaptation into practice of educatingpatients with type-1 diabetes mellitus to methodology of self-control ofglycemia and of technique of selection of adequate doses of administeredinsulin, a severe labile course of disease is noted in some patients. Anonset of frequent spontaneous (i.e. occurring without visible reasons)of hypoglycemic conditions often interchange by development of ketosis;and all attempts to reach metabolic compensation in these inpatientsrender only short-time relief. However, in 2-3 months afterxenotransplantation of cultures of islet cells, practically in all casesthe course of labile diabetes mellitus acquire more controllable andmanageable character. At the same time, usually happens stabilization ofindexes of carbohydrate metabolism and eradication of disposition toketosis.

Apparently, stabilization of labile forms of diabetes mellitus andrealization of compensation of carbohydrate metabolism are conditioned,first of all, by secretion of insulin by transplanted beta-cells. Duringthe post-transplantation period, administered dose of exogenous insulin(usually reduced in comparison to pre-transplantation level), sort ofsecures basic requirement in this hormone. In turn, insulin secreted bytransplanted beta-cells goes to recipient's blood more likely incorrelation with fluctuations of the level of glycemia, by doing sofacilitating more stable course of disease. Because significantconcentration of human C-peptide are being located in 1-2 months afterxenotransplantation in part of recipients with lack of signs offunctioning of own beta-cells (C-peptide-negative), we may assume thatpatient's partially restored islet apparatus steps in the process ofregulation of carbohydrate metabolism. Owing to that, in patients withprevious labile course of diabetes hypoglycemic status becomes lessexpressive, and more often—completely disappears because transplantedand restored beta-cells stop extracting insulin in situations whereglycemia approaches level close to normal. It appears, thatstabilization of level of glycemia after transplantation of cultures ofislet cells is conditioned by restoration, to some extent, of feedbackmechanism between level of glycemia and secretion of insulin, whichfeedback was absent in patients with diabetes mellitus type-1 because ofcell death caused by autoimmune process in islands of pancreas.

It is also possible, that some role in stabilization of IDDM and indecreasing of requirements of recipient in exogenous insulin may beattributed to normalization of insulin receptors in peripheral tissuesafter Islet Cells culture transplantation.

Because there was no significant reduction of requirements in exogenousinsulin in considerable proportion of recipients with positive effect oftransplantation of islet cells on diabetic angiopathy, it seems likereduction of administered insulin dose cannot be considered as a main,and especially, as sole sign of effectiveness of transplantationtreatment. If we put a purpose of achieving full insulin-independence asan end in itself, this approach can be fraught with risk of developmentof clinical situations dangerous for patient. As our experiencedemonstrates, a single-stage administration of significantly enlargedportions of xenogenous cultures of islet cells consisting practicallyonly from beta-cells, can provoke development of grave hypoglycemicstatus; but frequent (every 1-1.5 month) repeated transplantations of“regular” portions into liver (through permanent catheter to portalvein) can, at the end, lead to hardly predictable overdose of amount oftransplanted beta-cells.

No adequate insulin production by beta-cells that happens, likely,because of its inability to secrete insulin on strict principle offeedback, may lead to development of serious hypoglycemic conditions,even upon withdrawal of insulin injections. As a result, emaciation ofglycogenic depots in recipients and genesis of glucose by the way ofglucose-neo-genesis leads to accumulation of ketonic bodies andketo-acidosis.

Stay and regress of late diabetic complications shall be considered aspractically more meaningful, of greater prognostic importance, and asmore achievable in reality result of transplantation of cultures ofislet cells.

Of special importance is an effect of xenotransplantation of cultures ofislet cells on specific for diabetes mellitus impairment ofvessels—angiopathy—as they namely are the main reason of loss of sight(diabetic retinopathy).

Effect Mechanism of Islet Cells Transplantation on Late DiabeticComplications.

It is known that formation of diabetic angiopathies is very possibleeven in cases of ideal compensation of carbohydrate metabolism(norm-glycemia, a-glucosuria, normal concentration of glycozylatedhemoglobin) achieved with the help of intensified insulinotherapy. Atthe same time, after transplantation of islet cells cultures, in spiteof retention of elevated concentration of glycozylated hemoglobin inblood of significant numbers of patients, in majority of cases we seeslowing-down of progress and partial regress of late diabeticcomplications. That's why positive effect of islet cells transplantationcannot be explained, namely, by improving of indexes of carbohydratemetabolism.

In patients with insulin-dependent diabetes mellitus complicated byretinopathy the severity of changes on eye fundus is growing assecretion of insulin by patient's own pancreas decreases. Excretion ofendogen insulin was judged, certainly, on concentration of C-peptidesecreted into patient's blood in equimolar to insulin amounts. Wesuggested that progression of retinopathy was governed not only byreduction in secretion of insulin, but also by decrease in concentrationof C-peptide. After allogenous or xenogenous islet cells culturestransplantation in recipient with insulin-dependency caused by death ofbeta-cells of own pancreas, transplanted beta-cells started to emit intopatient's blood C-peptide (sure, simultaneously with insulin) which hehad been deprived of during several years, when, as in the capacity ofso-called substitution therapy he was getting injections of insulinpreparations only. In addition, in majority of patients inpost-transplantation period partial regeneration of pool of therecipient's own beta-cells takes place (Skaletskyy N. N., and others,1994 {6}.), which, naturally, start secreting insulin and C-peptide. Asa result, years—lasting deficit of C-peptide in the body is corrected,and that deficit might have been responsible for development of latespecific impairment of vessels and nerves.

Assumption of physiological role of C-peptide is at odds withwidely-accepted opinion that main role of C-peptide is purelystructural, connecting, and presented in facilitation of foldingmolecules of pro-insulin in such fashion that disulphide bonds betweenaminoacid remains of A- and B-chains of insulin molecules formed; andthat C-peptide possesses no biological potential, and as such is aballast molecule in physiological context.

Our assumption is supported by Swedish researches (Wahren J et al., 1991{15]). They demonstrated that C-peptide render stimulating effect onutilization of glucose by organism of insulin-dependent diabetespatient, although inhibiting influence on production of glucose by liveris not excluded. Lengthy (4 week) administering of C-peptide to type-1Diabetes mellitus patients assured better glycemic control (judging fromconcentration of glucose in fasting blood and on content of glycozylatedhemoglobin. In comparison with patients treated only with insulin. Veryimportant is the data showing that administering of human C-peptide haspositive influence on late complications of insulin-dependent diabetesmellitus. As such, in patients with diabetic nephropathy their renalfunction is improving, which improvement is demonstrated in reduction ofexcretion of albumin and reduction in glomerular filtration; in patientswith diabetic retinopathy penetrability of hemato-retinal barrier movesupwards; patients with autonomous neuropathy has slowing down of cardiacrate on inspiration and expiration. In addition, under the influence ofadministering of C-peptide, blood flow in working skeletal muscles ofdiabetes mellitus patients improves. Mechanism of such angio- andneuro-protective effect of C-peptide is unclear. As physiological effectof C-peptide is realized through promotion of function of cell membrane,then, apparently, it pertains to activation of Na+K+-ATF (adenosinetriphosphoric acid)—connected with membranes of different cells.

In conclusion, it worth pointing out that islet cells transplantation intype-1 diabetes mellitus is a very important, but auxiliary method oftherapy of type-1 diabetes mellitus. Anti-diabetic treatment should beintegrated. Correct combination of reasonable diet, graded physicalexercise, adequate sugar-reducing therapy may be necessary for success.Application of organ transplantation of pancreas or grafting of isletsdetailed from pancreas, are fully justified in providing medical help topatients with diabetic nephropathy in its terminal stage when there is aneed for transplantation of an allogenous kidney. At the earlier stagesof kidney impairment, and also in cases of diabetic nephropathy,retinopathy (except terminal stages), use of xenotransplantation ofcultured islet cells generated from pancreases of newlborn rabbits maybe very effective.

Duly application of cultured islet cells transplantation in complex(integrated) treatment of type-1 diabetes mellitus may substantiallyaffect the prognosis of critical illness. Prophylactics and decelerationof secondary diabetic complications, achieved with the help of regularrepeated transplantation, can render not only medical but significantsocio-economic effect by prevention or pulling off disabilities indiabetic patients and by raising their life-expectancy and longevity.

Classical method of coloring of cells on Mallori and byaldehyde-fuchsine was used for revealing beta-cells. For more specificdetection of insulin-containing cells (beta-cells) we lately useimmune-fluorescent method of morphologic analysis. Below is thedescription of basic components and phases of this method.

Immune-Histochemical Coloring of Islet Cells Cultures of Pancreas ofNewborn Rabbits.

For beta-cells identification we use cultures received during incubatingof pancreatic micro-fragments in plastic cultural Petri dishes(Corning-Costar). Suspension of cultivated cells and cellular clustersis washed three times from growth medium with the help of warm solutionof phosphate buffer (PBS). Then, cultures are fixated with 0.5% Formalinsolution during 20 minutes at room temperature. Then, cultures areperfused with PBS solution with added fetal serum (cow) tillconcentration reaches 1%, and incubate it during 60 minutes at roomtemperature for blockage of nonspecific sorption of antibodies. After3-time cleansing with PBS solution, mouse monoclonal antibodies toinsulin (Sigma) diluted by PBS solution with 5% of fetal cow serum, arelaid on cultures. After that Petri dishes with studied cultures areincubated for 120 minutes at room temperature. Then, 3-time cleansing inPBS and 2^(nd) antibodies are laid on cultures (anti-mice antibodiesmarked FITC), sustaining 45-minute exposition. After that, cultures arethrice cleansed in PBS. Then, 60% solution of glycerin and PBS is laidon cultures, and put it under cover glass. Ready preparations areexamined with fluorescent microscope and are photographed by digitalcamera.

Performed series of immune-histo-chemical analysis of islet cellscultured from pancreases f newly-born rabbits demonstrated that share ofinsulin-containing cells in cultures comprises from 78% to 90%(average—82.2%). Percent was calculated by counting cells in itsanalysis in faze contrast and then upon comparative analysis inluminescent microscope. In addition to islet cells, singular fibroblastsare found in the culture but their share, usually not exceeds 1-5%.Cells of epithelial origin are usually remaining cells in culture andmake 5-17%, which fact the immune-histo-chemical coloring (withmonoclonal antibodies against protein CytoKeratin 18) confirms; but theyare not beta-cells, as these cellular structures are not revealingpresence of insulin. Probably, they are just other types of islet cellswhose presence in the culture shall be considered highly physiologicalas they (alpha-cells, delta-cells, pp-cells) are natural surroundingsfor beta-cells, who in normal conditions comprise, to some extent, anautonomous morpho-physiological structure—Langerhans islands.

In addition to studying the cellular composition of the culture, wedetermined total amount of cells in it. By the way of methodicalcounting in more than 20 cultures, which calculation was performed atthe time of analysis under inverted microscope (Nikon, Japan) wesucceeded to determine that culture received out of 20 pancreases of1-2-day-old rabbits contains from 451600 to 568900 islet cells(average—521,500). One dose of Islet Cells Culture represents a fusionof 4 cultures of islet cells received from 80 pancreases of 1-2-day-oldrabbits. Amount of islet cells containing in such a dose is, in averagenot less than 2,000,000, at least 80% of which are beta-cells.

Cultures, produced through the above-described method are not cellularpreparation with strictly determined quantitative characteristics, suchas precise percentage of beta-cells in each culture. This is not apreparation as a result of strictly regulated chemical synthesis orgene-engineering manipulations. Cellular preparation used fortransplantation, represents a fusion of parallel grown cultures of isletcells, which, naturally are distinctive from each other to some degree.Analysis of cell composition, mainly rendering an idea of share ofbeta-cells in the culture demonstrated that it makes, for example, about80-94% while viability of these cells is from, for example, about 77% to85%. For biologic preparation this variation in figures is, in ouropinion, not significant. Upon increase of amount of cultures used for 1transplantation this variation may be reduces.

Immediately prior to a planned clinical transplantation (transportation)of islet cells culture, there is a selection of cultures with emphasison longevity of its cultivation, results of microscopic observation, andexpress-analysis for sterility and viability. Gathering cultures fortransplantation is conducted in conditions of laminar box securingsupply of constantly circulating sterile air. Selected cultures—at 4cultures for one transplantation dose is gathered with the help of aspecial cellular scraper (Cell scraper, Corning-Costar). Gatheredcellular suspension is being centrifuged in special 50-milliliterplastic test-tubes (800 rotations per minute for 10 minutes). Thencellular sediment is transferred to a sterile plastic 15-mm test tubeand is being suspended in a salt Hank's solution. Marked test-tube withcellular preparation is then placed into 50-mililiter test-tube andtightly closed with a lid additionally fixating it with a special film(Parafilm).

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

Example 15 Effect of Transplantation of Cultures of Islet Cells onCourse of Experimental Diabetes Mellitus in Rats, Subjected to Diet withVarious Protein Content, and Status of Pancreatic Islets in ExperimentalAnimals

Experiments were conducted on male-rats of Wistar line with initial bodymass of 100-120 grams. After a short adaptation period of staying on avivarium ration, 60 animals with body mass not less than 160 grams wereadministered sub-cutaneously an Alloxan in a dose of 200 mg/kg for thepurpose of induction of Diabetes Mellitus. After that, the rats weredivided into three equal groups (20 animals each) and were transferredto synthetic low-calorie ration with normal (18%—I group), lowered(9%—II group), and heightened (50%—III group) protein content (soyisolate Ardex M). At that, iso-caloric content of food ration of IIgroup rats was secured by increasing of quota of fats while preservingcarbohydrates quota.

In two weeks after administering Alloxan and commence of dietaryfeeding, 24 animals with severe and consistent diabetic status (majorityof animals showed glycemia of more than 20 mmol/l) were administeredxenotransplantation of CC cultures of pancreases of newborn rabbits. 8rats from each group became recipients of xenotransplantation.Suspension of cultivated CC in Hanks solution was injected into spleenpulp of animals by syringe through injection needle. In two weeks afterintra-spleen xenotransplantation of cultures, a very certain (P<0.05)glycemia decrease was noted in all three groups of rats-recipients, butit appeared to be lower expressed in the II group of animals (20.4 2.4to 15.0 3.1 mmol/l), and to be the most expressed in animals of the IIIgroup (from 20.3 3.5 to 12.0 2.6 mmol/l). At the same time, in controlgroups of animals placed on various protein-content diet but notsubjected to xenotransplantation of CC cultures, the glucose level didnot altered significantly during the whole course of this observation,excluding rats kept on ration of 50% protein (a spontaneous decrease inglycemia happened from 19.8 1.9 to 17.1 1.9 mmol/l). Upon histologicalexamination of spleens with implanted CC cultures, we were able toreveal a non-damaged CC in the I group in 6 of 8 animals, in II group—in3 out of 8 animals, and in III group—in all animals. There was not evenone occurrence of signs of lymphocytic infiltration of a transplant. Itseems, that diet with heightened content of protein affects favorablythe course of experimental Diabetes Mellitus in rats, and afterxenotransplantation of CC cultures, a ration of 50% protein guarantees amore expressed and permanent sugar-lowering effect of transplantation inrats-recipients.

In the same series of experiments, a morphologic status of pancreaticislet cells was studied in rats-recipients and in control animals.

The demonstrated method can also be applied to other mammals for thetreatment of Diabetes Mellitus.

Pancreases of 8 rats-recipients subjected to successfulxenotransplantation of CC cultures of pancreases of newborn rabbits,were examined histologically in 4 weeks after transplantation. For thatpurpose, pieces of organs were fixated in Bueno mix and sluiced in wax.Slices of 5-7 mkm were colored by hematoxilin and eosine, and also byAldehyde-Fuxin for detection of B-cells. At the same time, pancreases of6 control animals with untreated alloxan-induced Diabetes were examined,as well as 6 healthy rats that did not receive alloxan.

In studying pancreases of healthy intact rats, as expected, inLangergants islets had 45 to 76% of B-cells. Rats with untreatedalloxan-induced Diabetes, content of B-cells in preserved islets wassignificantly decreases, but the level of decrease, probably, dependedon protein content in animals' ration. As such, if in untreated animalson the I and III groups, share of B-cells in islet was, respectively,6.2 1.7 and 8.3 1.1%, but in the II group islets with at least sporadicB-cells was revealed only in 2 out of 6 rats. Significantly more highcontent of B-cells in islets was noted in rats-recipients. As such, in 4out of 8 animals of the I group that were subjected to intra-spleenxenotransplantation of CC cultures, in their own pancreases were foundtypical B-cells, and its share among islet cells was from 7 to 21% (inaverage 13.0 2.1%). In half of rats-recipients of the II group, inpancreases were found islets with β-cells content of 7 to 17% (inaverage 12.2 1.4%), as almost the same as in islets of pancreases ofanimals of the I group. Among rats-recipients of the III group, isletswith normal B-cells were already from 10 to 55% (in average 23.5 8.8%).So, the biggest pool of B-cells, restored under the influence ofxenotransplantation of CC cultures of newborn rabbits, was revealed inpancreases of rats-recipients that received ration with increasedprotein content.

In connection with completed experiments, it is possible to decide, thatanti-diabetic effect of xenotransplantation of cultures of islet cellson the course of experimental diabetes in rats is carried into effect bytwo ways: a. functioning of transplanted β-cells, confirmed in additionto expressed sugar-lowering effect of transplantation, by finding groupsof transplanted islet cells in spleen's pulp of animals-recipients;b.—stimulating effect of transplantation of islet cells culture at isletapparatus of pancreases of rats-recipients, which data of histologicalexamination witnesses to significantly higher frequency of locatingislets with normal beta-cells and bigger share of it in islets ofpancreases of rats-recipients than in rats with untreatedalloxan-induced diabetes.

Example 16 Demonstration of Angio-Protective Effect of Transplantationof Cultures of Islet Cells on the Sample of Experimental DiabeticNephropathy

The aim of the completed experimental research was studying of an effectof xenotransplantation of CC of pancreases on the course of diabeticnephropathy.

With the help of intra-abdominal injections of Streptozotocin (daily inthe dose of 10 mg/kg in the course of a week) Wistar Line rats werecaused to get diabetes mellitus. In 2-3 months after manifestation ofdiabetic status, 24 animals were selected with expressedmicro-albuminuria, and divided them in 3 groups. Rats of the I group (8animals) were performed a intra-abdominal transplantation of CC culturesreceived from pancreases of newborn rabbits using an original method.Each recipient was administered 300000-50000 of CC. Eight animals fromthe II group were treated by insulin-therapy in dosages adequate to itslevel of glycemia. The III group was comprised of 8 rats with untreatedexperimental Diabetes Mellitus. Glycemia was measured every week, aswell as micro-albuminuria.

Results: 7 out of 8 rats of the I group in 1-2 weeks aftertransplantation of CC cultures had decrease of their glycemia levels upto a normal or almost-normal level that stayed decreased until the endof observation term (12 weeks). At that, a significant decrease insecretion of albumines with urine was noted, with up to an almost normallevel in 4 recipients. At the same time, all animals of the II groupthat was subjected to insulin-therapy, had a steady increase of albumineconcentration in urine. Control group rats (without treatment ofdiabetes) a high level of glycemia persisted during the term of thewhole experiment, and micro-albuminuria increased up to a level of ahigh proteinuria. In addition, 3 animals fro control group had diabeticcataract not revealed in animals of the I and II groups.

On the grounds of obtained results, we made a conclusion thatXenotransplantation of CC cultures provides therapeutic effect on thecourse of experimental diabetic nephropathy, which, probably, related toangio-protecting effect of C-peptide secreted by transplanted CC.

It worth noting, that expressed and prolonged antidiabetic effect oftransplanted CC cultures from human fetuses, and also fetuses andnewborn animals, was achieved without application of immuno-isolation ofcell transplants, and without application of immune-suppressive therapy.It was sufficiently proved that CC's survivorship in organisms of alienanimals-recipients was provided by long-term cultivation of donorpancreatic tissue. And such conditions of incubation were created(temperature and gas regime of cultivation, special formula of culturalmedia), and death and elimination of immunologically aggressive exocrinetissue of pancreases, and also of those cellular elements (endothelium,so-called passenger-leucocytes) that are capable to initiate a rejectionof xenogenic cells. The most important is a lack of xenogenic vascularendothelium in the cellular transplant. As carbohydrates structuresexpressed on the latter, are the main target of previous, or naturalantibodies responsible for initiation of super-acute rejection ofxenotransplant.

For the purpose of studying an immunogenicity of cells comprisingcultures obtained from pancreases of newborn rabbits, at the laboratoryof Transplantation Immunology of N.I.I. (Scientific Research Institute)of Transplantology and Artificial Organs (Abramov V.U., Bogdanova N.B.),there were experiments conducted in defining of fixation of CC ofimmunoglobulin of human blood serum. Cells, incubated with differentserum of human blood, were dyed with monoclonal antibodies against humanimmunoglobulin, and were analyzed at the currency cytometer. As itappeared, the cells comprising the culture are capable of fixatingimmunoglobulin M on its surface, but at that we did not find anyfixation of immunoglobulin G. Probably, a fact of fixation by cultivatedislets of Immunoglobulin M does not have any significantimmune-pathogenic meaning, as different terms of cell incubation withserum and various concentrations of human complements did not lead toheightened (in comparison with control) lyses of islet cells.

Successful experimental research made grounds for carrying out clinicaltransplantation of cultures CC of pancreases of fetuses and newborn topatients with Diabetes Mellitus of Type I.

Since 1999, the main source of cultures is pancreas of newborn rabbitssupplied by special Vivarium RAMN (Russian Academy of Science).Selection of this donor material was supported by information thatrabbit's insulin is very close to human insulin in its structure, andalso an opportunity for abundant harvesting of such animals. It is alsovery important, that there has been no data in world rabbit-farmliterature about any possibility to spread zoonosic infections fromrabbits to humans. This information allows excluding, practically,spread of viruses and other infectious agents from rabbits to humansalong with transplanted cells, especially considering that immune systemof a recipient, while not suppressed by immune-suppressive therapy, iscapable to destroy those xenogenic infectious agents that,theoretically, can get to humans at the time of transplantation ofanimal cells.

Example 17 General Results of Xenotransplantation of Cultures of IsletCells of Pancreases to Rats with Induced Diabetes Mellitus

Ability of Islet Cells Cultures (CC) produces from pancreases of humanfetuses, and also if fetuses of newborn animals, to survive and functionin conditions in vivo, was demonstrated by us in experiments inxenotransplantation of such cultures to animals with experimentalDiabetes Mellitus.

Male rats of Wistar Line of body weight of 180-220 grams were used asexperimental animals, which were kept on usual vivarium feed and also(in the merits of special experiment) on the ration of various proteincontent.

Experimental Diabetes Mellitus in rats was induced by subcutaneousintroduction of Alloxan Solution in the dosage of 200 mg for 1 kilogramof a body mass, or by intra-abdominal introduction of StreptozotocineSolution in the doze of 60 mg/kg.

During experiments (both in regular and in control ones) there were usedonly rats with Diabetes induced by Alloxan or Streptozotocine, whoseGlycemia in fasting was 20 mmol/liter and more after two weeks fromadministration of diabetes-inducing preparations. Earlier conductedexperiments demonstrated that such animals do not exhibit a spontaneousreversion of experimental Diabetes Mellitus. 88 out of 104 rats withstable or critical Alloxan Diabetes (almost in 85%), aftertransplantation of Islet Cells Culture (CC), exhibited a firm remissionof diabetic status prior to the end of an experiment (20 weeks).

Antidiabetic effect of xenotransplantation was clearly demonstrated asupon administration of cultures into liver (through portal vein ordirectly into parenchyma of the organ), into spleen (cultures wereintroduced intro-pulpar), as well as into muscles of the anteriorabdominal wall. In rats with remission of experimental diabetes, in theplaces of implantation there was revealed an CC with preserved structureand signs of secretor activity even after 8 weeks after xenogenictransplantation.

In a series of research we demonstrated an ability of cryo-conservationof CC of pancreases of human fetuses and restoration of its vitalityafter defrosting and re-cultivating. We also conducted experiments incalibration of functional activity of defrosted cultures of CC of PZh ofhuman fetuses in in vitro conditions. For that purpose, CC—defrosted andcleansed from cryo-protector (glycerin or dimetilsulfoxid) obtained frompancreases of 4 human fetuses, were transplanted to 8 adult rats (bodymass 200-220 gr) with experimental (alloxan-induced) Diabetes Mellitus,who had their glycemia on the eve of transplantation (2 weeks before)was 24-32 mmol/l (in average 27.4 mmol/l). Suspension was introduceddirectly into parenchyma of liver of rats, the place of injection wascovered by 1-2 drops of medical glue (MK-6). 6 animals out of 8 duringthe 1-2 weeks after xenotransplantation showed decrease in theirglycemia to the level of not more than 15 mmol/l, and by the end of themonth a glucose concentration in the animals' blood was from 11 to 16mmol/l (in average 13.1 2.2 mmol/l). At that, clinical signs of DiabetesMellitus were vanishing (loss of body mass, loss of hair, polydypsia,polyuria). Remission of diabetic status kept during the wholeobservation period (5-7 weeks).

In a special series of experiments, a role of preliminary cultivation ofCC in vitro was demonstrated in its survivorship in a xenogenicrecipient's organism. For that purpose, a comparative analysis ofresults of xenotransplantation of CC of pancreases of human fetuses andxenotransplantation of non-cultivated fetal islet tissue to rats withexperimental Diabetes Mellitus. A significantly more expressed effect oftransplantation of pre-cultivated CC in contrast to transplantation ofnon-cultured pancreatic tissue that caused only short-term remission ofdiabetic status. As such, by the way of experiment, there was confirmedan immune-modulating effect of cultivation in vitro leading tosignificant increase in term of its survival in an organism of an alienrecipient.

Similar results were obtained in experimental xenotransplantation ofislet cell cultures produced from pancreases of fetuses of pigs andcattle, and also from newborn rabbits.

In experiments of intra-spleen transplantation of cultures of isletcells obtained from pancreases of newborn rabbits, there were twoscientific goals set. First one is aimed at studying of influence ofdiet with various content of protein on the course of experimentalDiabetes Mellitus in rats, and severity of sugar-lowering effect oftransplantation of pancreatic islet cell culture. Second goal, neverearlier addressed, was studying of effect of successfulxenotransplantation of CC cultures on status of own islet apparatus ofpancreases of rats-recipients.

Example 18 Clinical Transplantology of Islet Cells Cultures to Patientswith Diabetes Mellitus

Enormous demand in transplantation of islet cells cultures to patientswith Diabetes Mellitus of Type 1 upon obvious shortage of allogenicdonor materials (human embryos), forced us to use xenogenic islet cellscultures for clinical transplantations. Basics for such transplantationswere successful xeno-transplantations of islet cells cultures producedfrom pancreases of newborn rabbits to rats with experimental DiabetesMellitus. Applications of xeno-transplantations allow us, thanks toavailability of donor materials, to widely conduct repeated islet cellculture transplantations.

In addition, results of newborn rabbits' islet cells culturestransplantations to animals with experimental diabetic nephropathy, alsoshowed very successful results.

1. Clinical Transplantology of Islet Cells Cultures Produced fromPancreases of Newborn Rabbits.

Total transplantations performed: 3,500.

Below, are the results of a long-term (longer than 5 to 20 years)dynamic observations of 1,200 patients with Type 1 Diabetes.

There were 680 men and 520 women in the pool of 1,200 patients.Patients' age at the moment of transplantation was from 16 to 42 yearsold, average—28.1 years old.

It is known that the severity of manifestation of secondary diabeticcomplications depends very significantly on the longevity of thedisease. Supposedly, the death of own (islet cells) happens at the3-5^(th) year after manifestation of the disease. Secondary diabeticcomplications demonstrate itself usually in patients with history of thedisease of more than 10 years. Because of that, all patients weredivided into 3 groups with respect to longevity of the disease:

a. from 2 to 10 years—124 patients;b. from 11 to 20 years—344 patients;c. 20 years or more—732 patients.

Also, repeated transplantations with history of the disease of 20 yearsand more were performed on 267 patients;

9-10 transplantations with intervals of 6-11 months were performed to 11patients;7-8 transplantations were performed to 26 patients;5-6 transplantations were performed to 33 patients;3-4 transplantations—to 80 patients;2 transplantations—to 117 patients.Patients were examined to determine the character of Type 1 Diabetesdevelopment and to reveal complications.

For transplantation to one patient, we usually used islet cells culturesproduced from 3-40 pancreases of 1-2 day newborn rabbits. Emulsion ofislet cells culture collected before transplantation was introduced, asa rule, into straight abdominal muscle under local anesthesia. We didnot use immune-suppression.

Islet Cells Cultures Transplantation Technique

One dose of the islet cells culture is a sterile cellular emulsion insaline Hanks solution of 10-15 ml, and it is kept in plastic test-tubesspecially marked.

Using injection needle of no less than 7 cm in length and not less than1 mm in diameter, the content of test-tube fills the syringe, and isbeing administered into the straight abdominal muscle under localanesthesia. Injection spot is to be closed with sterile band.

Below, there is a list of results of xenotransplantation of islet cellscultures and its effect on Type 1 Diabetes development features, and onthe level of compensation of flawed metabolism and its level ofmanifestation in patients with various history of disease.

It is important to note, that we did not have a goal to, at any extent,normalize the content of glycated hemoglobin, which is being stronglyrecommended by official science of diabetes. Sharp decrease of glycemiclevel is dangerous with severity of vascular complications, so, with thehelp of islet cells cultures transplantations and moderateinsulin-therapy, we tried to secure gradual improvement of compensationof carbohydrate metabolism, and to evaluate post-transplantation changesin manifestation of late diabetic complications.

1.1. Transplantation of Islet Cells Cultures of Pancreases of NewbornRabbits to Type I Diabetes Mellitus Patients with History of Diseasefrom 2 to 10 Years.

Total patients under observation: 124 of Type 1 Diabetes history of 2 to10 years. 75 men and 49 women. Age of patients at the time of firsttransplantation: from 18 to 43 years (average 24.3 years).

16 out of 124 patients of this group had strong labile character ofdisease course. At such, 9 patients were noted for frequent (severaltimes a week) spontaneous (i.e. manifesting without obvious reasons)hypo-glycemic states, which was repeatedly treated inpatient withoutachieving stabilization of the course of disease and withoutsuccessfully selecting an adequate dosage of administered insulin. 4patients had labile Type 1 Diabetes with propensity to developing asevere case of ketosis; attempts to achieve metabolic compensation ofthese patients rendered only temporary effect.

27 patients (11 of them—with labile character of diabetes) revealedsymptoms of sensor-motoric neuropathy—parenthesis and throbbing pain inankle muscles usually at night time. 12 patients revealed an onsetclinical stage of diabetic nephropathy (stage 3 on Mogensen), 5patients—expressed stage of nephropathy (stage 4 on Mogensen), and 11patients—non-proliferative retinopathy.

After intramuscular xenotransplantation of islet cells cultures,majority of recipients during 1-2 months noted decrease of previouslysignificantly increased daily average level of glycemia. Stability ofits indexes in rates corresponding to satisfactory or good compensationof carbohydrate metabolism was further noted during, at least, 12 monthsof post-transplantation observation. As such, in 15 out of 16 patientswith labile Type 1 Diabetes, the course of disease had a stablecharacter: propensity to hypo-glycemic states eliminated, as well aspropensity to ketosis. Bettering of glycemic control aftertransplantation of islet cells cultures of pancreases of newborn rabbitsconfirms data of glycated hemoglobin in recipients' blood (drop frompre-transplantation 11.0% to 9.1%; to 8.7% and to 9.6% respectfully in6, 9 and 12 months after transplantation).

Increase in compensation levels of Diabetes Mellitus and clear tendencyto lowering of daily average level of glycemia, allowed to lessen doseof applied insulin by the end of 1-2 months period (in average for 12%)that remained relatively reduced in 3, 6, 9 and 12 months aftertransplantation—respectfully for 20.5%, 22.2%, 18.5% and 11.9%.

At such, all 27 patients with sensor-motoric neuropathy, in 1 to 3months after transplantation of islet cells cultures, had nocharacteristic clinical signs of this diabetic complication. 7 out of 12patients with onset stage of nephropathy had excretion of albumins withurine normalized (became 30 ml per day less); 4 out of 5 patients withexpressed nephropathy had micro-albuminorrhea decreased up to the levelof an introductory stage of such complication. All 11 patients withdiabetic retinopathy did not demonstrate worsening of eye down state,and 4 of them demonstrated decrease in retina edema, and lower amount ofmicro-aneurisms and new vessels.

Those were the changes noted during the 12 month period after firsttransplantation to Type 1 Diabetes patients with history of the diseasefrom 2 to 10 years.

Later, 87 patients of this group were administered repeatedintramuscular transplantations of islet cells cultures produced ofpancreases of newborn rabbits:

12 patients—7 transplantations,21 patients—5-6 transplantations,41 patients—3-4 transplantations,13 recipients—2 transplantations each.

No one of these patients further revealed local or general signs ofrejection of transplant or any allergic reaction after repeatedtransplantations. Repeated transplantations allowed to significantlypreserving a positive effect of the first islet cells culturestransplantation during the whole period of observation. And no onepatient demonstrated any signs of progression of secondary complicationsof Type 1 Diabetes, such as neuropathy, retinopathy, or nephropathy.

1.2. TRANSPLANTATION OF ISLET CELLS CULTURES OF PANCREASES of NewbornRabbits to Type I Diabetes Mellitus Patients with History of Diseasefrom 11 to 20 Years.

Under observation there were total of 731 patients with Type 1 Diabeteswith history of disease from 11 to 20 years. 354 of them were men, and377—women. Age of patients at the time of first transplantation: from 18to 49 years (average age—2.3 years).

37 patients had a very labile character of disease that could not bestabilized with repeated inpatient treatments.

568 patients had signs of secondary diabetic complications: 119patients—neuropathy (88—only sensor-motoric form and 31—together withautonomous neuropathy, onset stage of nephropathy (stage 3 onMogensen)—204 patients. Patients with chronic kidney insufficiency(stage 5 on Mogensen) were not accepted for islet cells culture due tolack of perspective. 361 patients demonstrated signs of diabeticretinopathy: non-proliferative stage in 190 patients, pre-proliferativestage—in 213 patients, and 42 patients with proliferative stage.

In 1-3 months after xenotransplantation, in 32 patients with labile Type1 Diabetes the course of the disease acquired a more stable andcontrolled character. At that, as a rule, a stabilization of indexes ofcarbohydrates metabolism was noted. Improvement of compensation ofmetabolism was confirmed by changes in content of glycated hemoglobin inthis group patients' blood: from 12.0% (average index) beforetransplantation, its level in three months already reduced to 10.8%, in6 months—to 9.4%, in 9 months—slightly increased to 10.9%, and by theend of year—decreased to 9.8%.

At the same time, these patients had a forced reduction of the dailyadministered insulin dose in comparison to pre-transplantation levels:in 3 months after transplantation in average for 12.2%, in 6 months—for19.1%, in 9 months—for 17.0%, and in 12 months—for 12.4%.

During post-transplantation period, signs of a more benign developmentof secondary complications were observed in this group's recipients.Symptoms of both sensor-motoric as well as autonomous neuropathy beganto show reduction already by the end of 1-1.5 month period aftertransplantation; and by the 3^(rd) month these signs practically stoppedbothering almost half of such patients. Also, majority of patients withdiabetic nephropathy by the end of 2-4 months after xenotransplantationdemonstrated significant improvement in the course of such dangerouscomplication. So, 99 out of 112 patients with onset stage revealedsignificant reduction of loss of albumin with urine, and 68 patientsshowed normalization of micro-albuminorrhea. 157 patients out of 204with expressed stage of this complication had proteinuria lowered, andin 39 patients there happened a return to micro-albuminorrhea stage. Atthat, practically all recipients with arterial hypertension noted stabletendency to normalization of arterial pressure indexes. Patients withdiabetic retinopathy had lesser positive effect: out of 213 patientswith pre-proliferative stage, a regress of pathologic changes of theretina was only noted in 31 patients. However, in the group ofrecipients with non-proliferative retinopathy as well as withpre-proliferative retinopathy, there was noted no worsening of clinicalstate of the eye down. A definite stabilization of the clinical coursealso happened in the most difficult group of patients—with proliferativeretinopathy. Only 7 out of 42 recipients showed further progression ofdiabetic changes in retina.

Repeated transplantations (in 6-12 months after the firsttransplantation) were administered to 541 patients: 7-8 times to 75patients; 5-6 times to 167 patients, 3-4 times to 122 patients, twice—to150 patients. In majority of cases, repeated transplantation of isletcells cultures, at a minimum, provided preservation of positive dynamicsin patients' state that had happened after the first transplantation. Atthat, in some cases a summation of remedial effect occurred. As anexample, we would like to demonstrate a result of concurrentlyadministered (with 7-9 month interval) 5 (five) transplantations to apatient with an advanced sensor-motoric neuropathy, that caused thispatient with history of disease of 8 years to loose workability due tosevere pain in extremities and expressed muscular atrophy, especiallylower extremities' muscles. After the first transplantation, pain in hislegs weakened, and after the second transplantation the pain completelydisappeared, and a volume and tonus of muscles began to restore. As aresult of performed transplantation, the muscular mass of the recipientenlarged during 4.5 years for 23 kilograms, muscular tonus normalized aswell as transmission of nerve impulse along motor nerves. At that, thediabetes' development became stable, and a dose of administered insulindecreased almost for 50%. It is possible, that decrease in demand inexogenic insulin was conditioned to some degree by serious restorationof insulin secretion by own recipient's b-cells, as a rate of humanC-peptide in recipient's blood is 0.1 ng/ml in fasting, and 0.1 ng/ml(stimulated by sumsstakal) up to, respectfully 0.36 and 0.55 ng/ml bythe end of 4-year term of post-transplantation observation.

1.3. Transplantation of Islet Cells Cultures of Pancreases of NewbornRabbits to Type I Diabetes Mellitus Patients with History of Disease for20 Years and Longer.

Total patients in this group—345: 161 men and 184 women. Age of patientsat the time of first transplantation: from 25 to 56 years (average 33.4years). History of disease; from 21 to 37 years (average 25.1 years).

17 patients had a true labile development of diabetes: spontaneoushypo-glycemic states often were followed with episodes of keto-acidosis.

328 patients demonstrated secondary diabetic complications: 111 patientshad neuropathy, including 47 cases of sensor-motoric neuropathy, and 54patients had combination of sensor-motoric and autonomous neuropathy.275 patients were diagnosed with diabetic nephropathy, includingintroductory nephropathy (stage 3 on Mogensen) in 52 patients, andexpressed stage—in 223 patients. Diabetic retinopathy was diagnosed in24 patients, non-proliferative stage—in 18 patients,pre-proliferative—in 155 patients, and proliferative—in 61 patients.

All recipients with initially labile development of the disease has avery fast (during 1-3 months) stabilization of level of glycemia, and amore adequate selection of insulin-therapy was done.

In patients of this group, a decrease of average level of daily averageglycemia from 10.7 mmol/l before transplantation to 8.8 mmol/l in 3months; to 8.5 mmol/l—in 6 months, and with slight increase to 8.9mmol/l in 9 months, and to 9.1% mmol/l—in 12 months.

In accordance with changes in glycemia, afterwards, the content ofglycated hemoglobin in recipients' blood decreased in average from 11.1%before transplantation to 9.0% in 12 months after transplantation.

Increase of level of compensation in carbohydrate metabolism wasaccompanied by some decrease in demand of exogenic insulin in patients.By the third months after xenotransplantation, a dose of administeredinsulin was lowered in these patients in average for 11.2%, in 6months—for 21.6%, in 9 months—for 18.5%, and in 12 months—only for10.8%, which testified to return to demand in exogenic insulin topre-transplantation levels. Maximal decrease was noted in a womanpatient S. (age 26 years, history of Type 1 Diabetes −22 years,secondary complications: senor-motoric neuropathy, expressednephropathy, pre-proliferative retinopathy). Already by the end of 2′week after transplantation of islet cells culture produced frompancreases of newborn rabbits, a demand of administered insulin wasdecreased from 36 to 24 units/day, in 6 weeks—to 16 units/day, and in 10weeks—to 4 units/day, i.e. for 90%). At that, on the background ofstable state, a level of daily-average glycemia did not excess 9 mmol/l.Content of HbA1c in 4 months after transplantation decreased to 8.7% anddid not excess 9% on a stretch of at least 1.5 years. At that, asignificant decrease in severity of secondary diabetic complicationshappened.

Effect of transplantations on a level of severity of diabeticcomplications, in majority of cases depended on its type and clinicalstage.

So, if all patients with sensor-motoric neuropathy had a significantimprovement right after the first islet cells culture transplantation,the majority of patients with autonomous neuropathy had a positiveeffect only after the second or third transplantation.

All of 52 patients with the onset stage of diabetic nephropathy notedafter transplantation a decrease in micro-proteinuria, including 21patients—had it normalized. 187 out of 223 patients with expressed stageof diabetic nephropathy had their level of loss of protein with urine,and 67 of these patients had macro-proteinuria changed tomicro-proteinuria. After administering repeated transplantation of isletcells cultures to these recipients, there appeared grounds to referlevel of diabetic damage to kidneys to the onset stage of nephropathy.Improvement of filtration ability of kidneys was accompanied by loweringof arterial pressure and its normalization.

All patients with non-proliferative retinopathy demonstrated noprogression of characteristic pathological changes during the whole termof observation, with improvement of eye down picture in 5 recipients(elimination of retinal edema, decrease in amount of micro-aneurisms andnew vessels).

In majority of recipients with pre-proliferative retinopathy (in 130patients out of 155), there wasn't any progression in diabetic damage ofretina. At that, practically all patients, who had had regularlaser-coagulation prior to islet cells culture transplantations, had nomore need in continuing laser-coagulation therapy.

At the same time, during administration of repeated transplantations ofislet cells cultures to patients with proliferative retinopathy, notalways sensible to count for cardinal positive changes in pathologicallydamaged retina. Only 9 out of 61 patients demonstrated partial backwarddevelopment of proliferative process. At that, in 3 patients asignificant thinning of fibrous tissues that participated in retinadetachment, which lead to weakening of traction of retina and itspartial reattachment. This lead to definite restoration of lost visualfunctions. However, arrest of proliferative process and preservation ofexisting visual functions in 22 recipients for a period of severalyears, cold be counted as obvious positive result of repeatedtransplantation treatments.

CONCLUSION

Results of above-described repeated transplantations of islet cellscultures harvested from newborn rabbits, allow considering this type oftransplantation treatment to be an effective method of prophylactics andtreatment of secondary complications of Type 1 Diabetes, especially ofits onset and moderately expressed stages.

BIBLIOGRAPHY/LITERATURE

-   1. Volkov I. E., Skaletskyy N. N., Schenev S. V./Preliminary results    of xenogeneic transplantation of cultures of islet cells of pancreas    of rabbit to children with insulin-dependent diabetes    mellitus.//Bulletin of Experimental Biology & Medicine.—1998.—No 3.    Volume 126. P. 105-108-   2. Gavrilova N. A., Skeltskyy N. N./Effect of xenotransplantation of    pancreatic islet cells on pathogenic mechanisms of development and    course of diabetic retinopathy.//Reporter of Transplantology &    Artificial Organs.—2004.—No 1—P. 30-36.-   3. Skaletskaya G. N., Kirsanova L. A., Skaletskyy N. N., and    others./Change of course of experimental diabetic nephropathy under    influence of xenotransplantation of islet cells cultures. Materials    of the III All-Russian Conference on Transplantology & Artificial    Organs.//Reporter on Transplantology & Artificial Organs.—2005.—No    3.—P. 47.-   4. Skaletskyy N. N., Kirsanova L. A., Blyumkin V. N./Producing    cultures of islet cells of pancreases and its    transplantation.//Issues of Transplantology & Artificial Organs.—M.,    1994.—P. 73-80.-   5. Skaletskyy N. N., Shumakov V. I./Transplantation of islet cells    in treatment of diabetes mellitus//Transplantation of fetalissues    and cells./Bulletin of Experimental Biology & Medicine.—1998.—T.    126.—Suppl. 1.—P. 109-114.-   6. Shumakov V. I., Blyumkin V. N., Skaletskyy N. N., and others.    Transplantation of pancreatic islet cells.—M. Canon, 1995.—P. 384.-   7. Shumakov V. I., Skaletskyy N. N./Regulation of carbohydrate    metabolism and correction of impairment of carbohydrate metabolism    at diabetes Mellitus.//Essay on physiological problems of    transplantology and use of artificial organs/Under edition of    Academician V. I. Shumakov.—Tula: Repronix Ltd., 1998.—P. 93-118.-   8. Shumakov V. I., Skaletskyy N. N. Transplantation of islet and    other endocrine cells.//Transplantology (under edition of    Academician V. I. Shumakov).—M: Medicine, 1995.—P. 317-331.-   9. Gill R. G., The Immunology of Pancreatic Islet    Transplantation.—in book “Type 1 Diabetes”, Oxford University Press,    1996.—P. 118-133-   10. Shapiro A. M. J., Lakey J. R. T., Paty B. W., et al./Strategic    opportunities in clinical islet    transplantation//Transplantation.—2005.—Vol. 79.—P. 1304-1307.-   11. Shapiro A. M. J., Lakey J. R. T., Ryan E. A., et al./Islet    transplantation in seven patients with type 1 diabetes mellitus    using a glucocorticoid-free regimen//New England J. of    Medicine.—2000. V. 343.—P. 230-238.-   12. Wahren J., Johansson B.-L., Wallberg-Henriksson H./Does    C-peptide have a physiological role?//Diabetologia.—1994.—37, suppl.    2.—P. 99-107.-   13. Shumakov V. I., Skaletskyy N. N., Evseev Yu. N. et    al./Intraportal xenotransplantation of islet cell cultures in    diabetic patients//Biomaterial Living System Interactions.—1993.    Vol. 1.—N4.—P. 179-184.-   14. George S. Eizenbarth, Kevin J. Lafferty. Type I    Diabetes—Molecular, Cellular & Clinical Immunology.—Oxford    University press.

What is claimed is:
 1. A composition comprising neo-islet cells isolatedfrom rabbit pancreases, wherein the neo-islet cells are isolated by amethod comprising: harvesting said pancreases of newborn rabbits andplacing pancreases in a salt solution comprising an antibiotic at atemperature of 4-10° C.; removing vessels and excretory ducts from theharvested pancreases; obtaining minced pancreatic micro fragments fromsaid pancreases; transferring the minced pancreatic micro fragments intoa receptacle container, wherein the receptacle container is adapted fora rotating device; incubating the minced pancreatic micro fragments in aserum free medium at temperature 36° C. to 37° C. for 5 to 12 days at 0%to 5% CO₂ for a first incubation period, wherein the receptaclecontainer rotated by the rotating device at a constant speed of 2 to 6RPM; periodically replacing the serum free medium with fresh serum freemedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are beta-islet cells; and collectingneo-islet cells formed by association of free floating beta-islet cells.2. A composition comprising beta-islet cell clusters isolated fromrabbit pancreases, wherein the beta-islet cell clusters are isolated bya method comprising: harvesting said pancreases of newborn rabbits andplacing pancreases in a salt solution comprising an antibiotic at atemperature of 4-10° C.; removing vessels and excretory ducts from theharvested pancreases; obtaining minced pancreatic micro fragments fromsaid pancreases; transferring the minced pancreatic micro fragments intoa receptacle container, wherein the receptacle container is adapted fora rotating device; incubating the minced pancreatic micro fragments in aserum free medium at temperature 36° C. to 37° C. for 5 to 12 days at 0%to 5% CO₂, wherein the receptacle container rotated by the rotatingdevice at a constant speed of 2 to 6 RPM; periodically replacing theserum free medium with fresh serum free medium and removingspontaneously destroyed unwanted cells comprising exocrine cells andblood cells and elements of connective tissue until at least 80% ofremaining cells are beta-islet cells; collecting the beta-islet cellclusters; incubating the beta-islet cell clusters in a serum free mediumat temperature 18° C. to 28° C. for 4 to 10 days at 0% to 5% CO₂;periodically replacing the serum free medium with fresh serum freemedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are beta-islet cells; and collecting thebeta-islet cell clusters.
 3. A composition comprising progenic cells andbeta-islet cells isolated from rabbit pancreases, wherein the progeniccells and beta-islet cells are isolated by a method comprising:harvesting said pancreases of newborn rabbits and placing pancreases ina salt solution comprising an antibiotic at a temperature of 4-10° C.;removing vessels and excretory ducts from the harvested pancreases;obtaining minced pancreatic micro fragments from said pancreases;transferring the minced pancreatic micro fragments into a receptaclecontainer, wherein the receptacle container is adapted for a rotatingdevice; incubating the minced pancreatic micro fragments in a serum freemedium at temperature 36° C. to 37° C. for 5 to 12 days at 0% to 5% CO2,wherein the receptacle container rotated by the rotating device at aconstant speed of 2 to 6 RPM; periodically replacing the serum freemedium with fresh serum free medium and removing spontaneously destroyedunwanted cells comprising exocrine cells and blood cells and elements ofconnective tissue until at least 80% of remaining cells are beta-isletcells; collecting residual tissues of micro fragments; incubating theresidual tissues of micro fragments in 7% rabbit serum medium attemperature 36° C. to 37.2° C. for 5 to 10 days at 0% to 5% CO₂;periodically replacing the medium with fresh medium and removingspontaneously destroyed unwanted cells comprising exocrine cells andblood cells and elements of connective tissue until at least 80% ofremaining cells are progenic cells or beta-islet cells; and collectingthe progenic cells and beta-islet cells.
 4. A method of obtainingneo-islet cells isolated from rabbit pancreases, wherein the methodcomprising: harvesting said pancreases of newborn rabbits and placingpancreases in a salt solution comprising an antibiotic at a temperatureof 4-10° C.; removing vessels and excretory ducts from the harvestedpancreases; obtaining minced pancreatic micro fragments from saidpancreases; transferring the minced pancreatic micro fragments into areceptacle container, wherein the receptacle container is adapted for arotating device; incubating the minced pancreatic micro fragments in aserum free medium at temperature 36° C. to 37° C. for 5 to 12 days at 0%to 5% CO₂ for a first incubation period, wherein the receptaclecontainer rotated by the rotating device at a constant speed of 2 to 6RPM; periodically replacing the serum free medium with fresh serum freemedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are beta-islet cells; and collectingneo-islet cells formed by association of free floating beta-islet cells.5. A method of obtaining beta-islet cell clusters isolated from rabbitpancreases, wherein the method comprising: harvesting said pancreases ofnewborn rabbits and placing pancreases in a salt solution comprising anantibiotic at a temperature of 4-10° C.; removing vessels and excretoryducts from the harvested pancreases; obtaining minced pancreatic microfragments from said pancreases; transferring the minced pancreatic microfragments into a receptacle container, wherein the receptacle containeris adapted for a rotating device; incubating the minced pancreatic microfragments in a serum free medium at temperature 36° C. to 37° C. for 5to 12 days at 0% to 5% CO₂, wherein the receptacle container rotated bythe rotating device at a constant speed of 2 to 6 RPM; periodicallyreplacing the serum free medium with fresh serum free medium andremoving spontaneously destroyed unwanted cells comprising exocrinecells and blood cells and elements of connective tissue until at least80% of remaining cells are beta-islet cells; collecting the beta-isletcell clusters; incubating the beta-islet cell clusters in a serum freemedium at temperature 18° C. to 28° C. for 4 to 10 days at 0% to 5% CO₂;periodically replacing the serum free medium with fresh serum freemedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are beta-islet cells; and collecting thebeta-islet cell clusters.
 6. A method of obtaining a compositioncomprising progenic cells and beta-islet cells isolated from rabbitpancreases, wherein the method comprising: harvesting said pancreases ofnewborn rabbits and placing pancreases in a salt solution comprising anantibiotic at a temperature of 4-10° C.; removing vessels and excretoryducts from the harvested pancreases; obtaining minced pancreatic microfragments from said pancreases; transferring the minced pancreatic microfragments into a receptacle container, wherein the receptacle containeris adapted for a rotating device; incubating the minced pancreatic microfragments in a serum free medium at temperature 36° C. to 37° C. for 5to 12 days at 0% to 5% CO₂, wherein the receptacle container rotated bythe rotating device at a constant speed of 2 to 6 RPM; periodicallyreplacing the serum free medium with fresh serum free medium andremoving spontaneously destroyed unwanted cells comprising exocrinecells and blood cells and elements of connective tissue until at least80% of remaining cells are beta-islet cells; collecting residual tissuesof micro fragments; incubating the residual tissues of micro fragmentsin 7% rabbit serum medium at temperature 36° C. to 37.2° C. for 5 to 10days at 0% to 5% CO₂; periodically replacing the medium with freshmedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are progenic cells or beta-islet cells;and collecting the progenic cells and beta-islet cells.
 7. A method oftreating diabetes in a patient in need of such treatment byadministration of neo-islet cells isolated from rabbit pancreases,wherein the neo-islet cells are isolated by a method comprising:harvesting said pancreases of newborn rabbits and placing pancreases ina salt solution comprising an antibiotic at a temperature of 4-10° C.;removing vessels and excretory ducts from the harvested pancreases;obtaining minced pancreatic micro fragments from said pancreases;transferring the minced pancreatic micro fragments into a receptaclecontainer, wherein the receptacle container is adapted for a rotatingdevice; incubating the minced pancreatic micro fragments in a serum freemedium at temperature 36° C. to 37° C. for 5 to 12 days at 0% to 5% CO2for a first incubation period, wherein the receptacle container rotatedby the rotating device at a constant speed of 2 to 6 RPM; periodicallyreplacing the serum free medium with fresh serum free medium andremoving spontaneously destroyed unwanted cells comprising exocrinecells and blood cells and elements of connective tissue until at least80% of remaining cells are beta-islet cells; and collecting neo-isletcells formed by association of free floating beta-islet cells; andfurther wherein the neo-islet cells are administered to the patient totreat diabetes.
 8. The method of claim 7, wherein the patient has type Idiabetes.
 9. The method of claim 7, wherein the administration isintraperitoneal, parenteral, intravenous, intramuscular, subcutaneous,or intrathecal
 10. The method of claim 7, wherein the administration isinto liver through portal vein, directly into parenchyma of the liver,or into spleen.
 11. A method of treating diabetes in a patient in needof such treatment by administration of beta-islet cell clusters isolatedfrom rabbit pancreases, wherein the beta-islet cell clusters areisolated by a method comprising: harvesting said pancreases of newbornrabbits and placing pancreases in a salt solution comprising anantibiotic at a temperature of 4-10° C.; removing vessels and excretoryducts from the harvested pancreases; obtaining minced pancreatic microfragments from said pancreases; transferring the minced pancreatic microfragments into a receptacle container, wherein the receptacle containeris adapted for a rotating device; incubating the minced pancreatic microfragments in a serum free medium at temperature 36° C. to 37° C. for 5to 12 days at 0% to 5% CO2, wherein the receptacle container rotated bythe rotating device at a constant speed of 2 to 6 RPM; periodicallyreplacing the serum free medium with fresh serum free medium andremoving spontaneously destroyed unwanted cells comprising exocrinecells and blood cells and elements of connective tissue until at least80% of remaining cells are beta-islet cells; collecting the beta-isletcell clusters; incubating the beta-islet cell clusters in a serum freemedium at temperature 18° C. to 28° C. for 4 to 10 days at 0% to 5% CO2;periodically replacing the serum free medium with fresh serum freemedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are beta-islet cells; and collecting thebeta-islet cell clusters; and further wherein the beta-islet cellclusters are administered to the patient to treat diabetes.
 12. Themethod of claim 11, wherein the patient has type I diabetes.
 13. Themethod of claim 11, wherein the administration is intraperitoneal,parenteral, intravenous, intramuscular, subcutaneous, or intrathecal 14.The method of claim 11, wherein the administration is into liver throughportal vein, directly into parenchyma of the liver, or into spleen. 15.A method of treating diabetes in a patient in need of such treatment byadministration of progenic cells and beta-islet cells isolated fromrabbit pancreases, wherein the progenic cells and beta-islet cells areisolated by a method comprising: harvesting said pancreases of newbornrabbits and placing pancreases in a salt solution comprising anantibiotic at a temperature of 4-10° C.; removing vessels and excretoryducts from the harvested pancreases; obtaining minced pancreatic microfragments from said pancreases; transferring the minced pancreatic microfragments into a receptacle container, wherein the receptacle containeris adapted for a rotating device; incubating the minced pancreatic microfragments in a serum free medium at temperature 36° C. to 37° C. for 5to 12 days at 0% to 5% CO2, wherein the receptacle container rotated bythe rotating device at a constant speed of 2 to 6 RPM; periodicallyreplacing the serum free medium with fresh serum free medium andremoving spontaneously destroyed unwanted cells comprising exocrinecells and blood cells and elements of connective tissue until at least80% of remaining cells are beta-islet cells; collecting residual tissuesof micro fragments; incubating the residual tissues of micro fragmentsin 7% rabbit serum medium at temperature 36° C. to 37.2° C. for 5 to 10days at 0% to 5% CO2; periodically replacing the medium with freshmedium and removing spontaneously destroyed unwanted cells comprisingexocrine cells and blood cells and elements of connective tissue untilat least 80% of remaining cells are progenic cells or beta-islet cells;and collecting the progenic cells and beta-islet cells; and furtherwherein the progenic cells and beta-islet cells are administered to thepatient to treat diabetes.
 16. The method of claim 15, wherein thepatient has type I diabetes.
 17. The method of claim 15, wherein theadministration is intraperitoneal, parenteral, intravenous,intramuscular, subcutaneous, or intrathecal
 18. The method of claim 15,wherein the administration is into liver through portal vein, directlyinto parenchyma of the liver, or into spleen.